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.

Virus-Like Particles of SARS-CoV-2 as Virus Surrogates: Morphology, Immunogenicity, and Internalization in Neuronal Cells.

The engineering of virus-like particles (VLPs) is a viable strategy for the development of vaccines and for the identification of therapeutic targets without using live viruses. Here, we report the generation and characterization of quadruple-antigen SARS-CoV-2 VLPs. VLPs were generated by transient transfection of two expression cassettes in adherent HEK293T cells─one cassette containing Mpro for processing of three structural proteins (M, E, and N), and the second cassette expressing the Spike protein. Further characterization revealed that the VLPs retain close morphological and antigenic similarity with the native virus and also bind strongly to the SARS-CoV-2 receptor hACE-2 in an in vitro binding assay. Interestingly, the VLPs were found to internalize into U87-MG cells through cholesterol-rich domains in a dynamin-dependent process. Finally, our results showed that mice immunized with VLPs induce robust humoral and cellular immune responses mediated by enhanced levels of IL-4, IL-17, and IFNγ. Taken together, our results demonstrate that VLPs mimic the native virus and induce a strong immune response, indicating the possible use of these particles as an alternative vaccine candidate against SARS-CoV-2. VLPs can also be effective in mapping the initial stages of virus entry and screening inhibitors.

High-manganese and nitrogen stabilized austenitic stainless steel (Fe-18Cr-22Mn-0.65N): a material with a bright future for orthopedic implant devices.

The rationale behind the success of nickel free or with extremely low nickel austenitic high manganese and nitrogen stabilized stainless steels is adverse influences of nickel ion on human body. Replacement of nickel by nitrogen and manganese provides a stable microstructure and facilitates better biocompatibility in respect of the conventional 316L austenitic stainless steel (316L SS). In this investigation, biocompatibility of the high-manganese and nitrogen stabilized (Fe-18Cr-22Mn-0.65N) austenitic stainless steel was studied and found highly promising.In vitrocell culture and cell proliferation (MTT) assays were performed on this stainless steel and assessed in respect of the 316L SS. Both the steels exhibited similar cell growth behavior. Furthermore, an enhancement was observed in cell proliferation on the Fe-18Cr-22Mn-0.65N SS after surface modification by ultrasonic shot peening (USP). The mean percent proliferation of the MG-63 cells increased from ≈88% for Un-USP to 98% and 105% for USP 3-2 and USP 2-2 samples, respectively for 5 d of incubation. Interestingly,in vivoanimal study performed in rabbits for 3 and 6 weeks showed callus formation and sign of union without any allergic reaction.

The effect of amantadine on an ion channel protein from Chikungunya virus.

Viroporins like influenza A virus M2, hepatitis C virus p7, HIV-1 Vpu and picornavirus 2B associate with host membranes, and create hydrophilic corridors, which are critical for viral entry, replication and egress. The 6K proteins from alphaviruses are conjectured to be viroporins, essential during egress of progeny viruses from host membranes, although the analogue in Chikungunya Virus (CHIKV) remains relatively uncharacterized. Using a combination of electrophysiology, confocal and electron microscopy, and molecular dynamics simulations we show for the first time that CHIKV 6K is an ion channel forming protein that primarily associates with endoplasmic reticulum (ER) membranes. The ion channel activity of 6K can be inhibited by amantadine, an antiviral developed against the M2 protein of Influenza A virus; and CHIKV infection of cultured cells can be effectively inhibited in presence of this drug. Our study provides crucial mechanistic insights into the functionality of 6K during CHIKV-host interaction and suggests that 6K is a potential therapeutic drug target, with amantadine and its derivatives being strong candidates for further development.

A Dual Drug Delivery Platform for Cancer-Bacteria Cotargeting.

Bacteria can associate with mammalian cells in different ways. While some are essential for the body, others can manipulate their local environment to cause tumor-like conditions or find refuge in already existing cancerous tissues or cells, impacting not only chemotherapy through drug transformation but also antibiotic resistance through immune evasion. Despite these facts, cancer and bacterial therapies continue to be administered independently. We have developed a dual drug delivery platform, called "dualosome", that not only targets cancer cells but also clears bacteria from the cancer niche. Dualosomes comprise liposomes loaded with an anticancer drug (doxorubicin) in their core and a cationic antibacterial peptide (sushi S3) on their surface. Folic acid is also attached to the liposomal surface to impart cancer cell specificity. The efficacy of dualosomes is demonstrated on model S. typhi-infected hepatoma (Huh-7) cells, and it shows that the copackaged system is at least 75% more effective in eliminating both cell types than either drug alone, in the nanoformulated or free form. This improved performance is attributed to the bacteria-linked doxorubicin activity, as well as the enhanced internalization of the liposomes due to their cationic surface charge. Overall, our system holds great promise for curbing cancer-related bacterial infections and drug resistance in both cell types.

Breach: Host Membrane Penetration and Entry by Nonenveloped Viruses.

Disruption of host membranes by nonenveloped viruses, which allows the nucleocapsid or genome to enter the cytosol, is a mechanistically diverse process. Although the membrane-penetrating agents are usually small, hydrophobic or amphipathic peptides deployed from the capsid interior during entry, their manner of membrane interaction varies substantially. In this review, we discuss recent data about the molecular pathways for externalization of viral peptides amidst conformational alterations in the capsid, as well as mechanisms of membrane penetration, which is influenced by structural features of the peptides themselves as well as physicochemical properties of membranes, and other host factors. The membrane-penetrating components of nonenveloped viruses constitute an interesting class of cell-penetrating peptides, and may have potential therapeutic value for gene transfer.

Synonymous codon usage of genes in polymerase complex of Newcastle disease virus.

Newcastle disease virus (NDV) is pathogenic to both avian and non-avian species but extensively finds poultry as its primary host and causes heavy economic losses in the poultry industry. In this study, a total of 186 polymerase complex comprising of nucleoprotein (N), phosphoprotein (P), and large polymerase (L) genes of NDV was analyzed for synonymous codon usage. The relative synonymous codon usage and effective number of codons (ENC) values were used to estimate codon usage variation in each gene. Correspondence analysis (COA) was used to study the major trend in codon usage variation. Analyzing the ENC plot values against GC3s (at synonymous third codon position) we concluded that mutational pressure was the main factor determining codon usage bias than translational selection in NDV N, P, and L genes. Moreover, correlation analysis indicated, that aromaticity of N, P, and L genes also influenced the codon usage variation. The varied distribution of pathotypes for N, P, and L gene clearly suggests that change in codon usage for NDV is pathotype specific. The codon usage preference similarity in N, P, and L gene might be detrimental for polymerase complex functioning. The study represents a comprehensive analysis to date of N, P, and L genes codon usage pattern of NDV and provides a basic understanding of the mechanisms for codon usage bias.

Analysis of synonymous codon usage in the VP2 protein gene of infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is an important poultry pathogen. The VP2 protein of IBDV is the major host-protective immunogen. Although the functions of the VP2 protein have been well studied, the factors shaping synonymous codon usage bias and nucleotide composition in the VP2 gene have not yet been reported. In the present study, we have analyzed the relative synonymous codon usage and effective number of codons (ENC) using 69 IBDV VP2 genes. The major trend in codon usage variation was studied using correspondence analysis. The plot of ENC values and GC3s as well as the correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in the VP2 gene. In addition, other factors, such as the aromaticity, hydrophobicity and aliphatic index also influence the codon usage variation of the VP2 gene. This study represents a comprehensive analysis of IBDV VP2 gene codon usage patterns and provides a basic understanding of the codon usage bias.

Species based synonymous codon usage in fusion protein gene of Newcastle disease virus.

Newcastle disease is highly pathogenic to poultry and many other avian species. However, the Newcastle disease virus (NDV) has also been reported from many non-avian species. The NDV fusion protein (F) is a major determinant of its pathogenicity and virulence. The functionalities of F gene have been explored for the development of vaccine and diagnostics against NDV. Although the F protein is well studied but the codon usage and its nucleotide composition from NDV isolated from different species have not yet been explored. In present study, we have analyzed the factors responsible for the determination of codon usage in NDV isolated from four major avian host species. The F gene of NDV is analyzed for its base composition and its correlation with the bias in codon usage. Our result showed that random mutational pressure is responsible for codon usage bias in F protein of NDV isolates. Aromaticity, GC3s, and aliphatic index were not found responsible for species based synonymous codon usage bias in F gene of NDV. Moreover, the low amount of codon usage bias and expression level was further confirmed by a low CAI value. The phylogenetic analysis of isolates was found in corroboration with the relatedness of species based on codon usage bias. The relationship between the host species and the NDV isolates from the host does not represent a significant correlation in our study. The present study provides a basic understanding of the mechanism involved in codon usage among species.

Effect of lead and cadmium on aquatic plant Hydrilla verticillata.

Absorption of different concentrations of Lead (Pb) and Cadmium (Cd) by aquatic plant Hydrilla vertcillato was measured during winter season for two different durations i.e. 3 days and 7 days. Effect of Pb and Cd was evaluated by analyzing various parameters such as biomass, total chlorophyll, carotenoid, protein, nitrate reductase activity, SOD (super oxide dismutase) and heavy metal uptake. Increase in biomass, total chlorophyll, protein and nitrate reductase activity was noticed at lower concentration of both metals whereas at higher metal concentrations of Pb and Cd, decrease in these parameters was observed i.e. it was concentration and duration dependent. Increase in carotenoid and SOD levels at high concentration of Pb and Cd indicated its ability of stress tolerance. Accumulation of Pb by test plant was found to be more than Cd at low concentration. Higher concentration of Cd and Pb caused toxicity which resulted in reduced plant growth and physiological activities.

Microbial keratitis after descemet stripping automated endothelial keratoplasty.

BACKGROUND: To report two cases of corneal infection after Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: Two eyes of two patients demonstrated varying clinical presentations of microbial keratitis after DSAEK. At the initial presentation, the keratitis involved the host cornea alone in case 1, whereas in case 2, the posterior lamellar disk alone was involved. A pair of microvitrectomy scissors was used in case 2 from the side port to obtain a 2-mm sample of the posterior lamellar disk for microbiologic evaluation. The keratitis did not respond to medical therapy, and therapeutic penetrating keratoplasty was performed to resolve the infection in both the eyes. The main outcome measures were resolution of infection, absence of recurrence of keratitis, graft clarity, and visual outcome. RESULTS: There was complete resolution of infection after full thickness therapeutic grafts with best-corrected visual acuities of 20/60 and 20/40, respectively. CONCLUSIONS: Initial presentation of microbial keratitis after DSAEK may involve either the host or the posterior lamellar disk alone. A microvitrectomy scissors through the side port may be used for biopsy of posterior lamellar disk in recalcitrant infection.

Comparison of the efficacy and safety of different methods of posterior subtenon injection.

PURPOSE: To compare the safety and efficacy of different methods of posterior subtenon (PST) injection of corticosteroids in the treatment of cystoid macular edema secondary to intermediate uveitis. DESIGN: Prospective comparative randomized interventional study. PARTICIPANTS: A total number of 30 eyes with cystoid macular edema secondary to intermediate uveitis were examined. METHODS: Patients were randomized into 3 treatment groups of 10 eyes each. Each group received PST injection of triamcinolone acetonide 0.5 mL (20 mg) by one of three methods: cannula method (group 1), Smith and Nozik method (group 2), or orbital floor injection method (group 3). Patients underwent Snellen's and ETDRS visual acuity (VA) testing, clinical evaluation, optical coherence tomography (OCT), and fundus fluorescein angiography (FFA) at baseline and follow-up visits. OUTCOME MEASURES: Changes in Snellen and ETDRS VA, OCT retinal thickness and assessments of safety were recorded in follow-up visits. RESULTS: Postintervention patients were followed up at the 1st, 2nd, 6th, and 12th weeks. Statistically significant (p = .00) improvement in VA was present in group1 from 0.25 +/- 0.08 (mean +/- standard deviation) to 0.75 +/- 0.24, in group 2 from 0.29 +/- 0.12 to 0.78 +/- 0.23, and in group 3 from 0.24 +/- 0.10 to 0.72 +/- 0.27. Statistically significant decrease in OCT central macular thickness (43.97% in group 1, 32.46% in group 2, and 29.75% in group 3) was noted at 12 weeks. However, the difference between individual groups at each visit did not reach statistical significance. Steroid-induced rise in intraocular pressure was observed in all the three groups with no statistical difference between individual groups. CONCLUSIONS: The different methods of PST injection are equally efficacious in terms of improving visual acuity. However, the cannula method achieves the greatest quantitative reduction in macular thickness. As the cannula method is as efficacious as Smith and Nozik method it may be a preferable method to deliver posterior subtenon injection of corticosteroids.