Synergistic Effect of Zinc-Chitosan Nanoparticles and Hydroxychloroquine to Inhibit Buffalo Coronavirus.

Zinc ions can hinder the synthesis of proteins required for accomplishing several stages of the viral life cycle. The intracellular zinc concentration can be increased by using zinc ionophores which transport zinc ions into the cells and hinder viral replication. (Hydroxy)chloroquine is an example of a zinc ionophore, but both zinc and (hydroxy)chloroquine can be toxic to the host organism. The nanocarriers may serve as camouflage to evade the adverse effects of drugs, chemicals, and nanoparticles on the host. We formulated ZnO nanoparticles with flower-like morphology (ZnONFs). It was further decorated with chitosan along with hydroxychloroquine (as a zinc ionophore) (CHCZnO NPs). We have chosen the cationic polymer chitosan since it is biocompatible, biodegradable and binds easily with the cells, and enhances the transport of drugs across cell membranes. The formulation was investigated for size, shape, surface charge, and interaction of chemicals used. We evaluated the formulations for cytotoxicity, and biocompatibility in embryonated chicks and their efficacy against bovine coronavirus (BCoV) isolated from a buffalo calf, and pneumo-enteric coronaviruses isolated from a buffalo calf with promising results in comparison to ZnONFs/hydroxychloroquine alone. Furthermore, we elucidate the mechanism underlying the lysosomotropic effect of various formulations on Vero cells infected with the buffalo coronavirus.

Nanoformulation of a Trypanocidal Drug Isometamidium Chloride Ameliorates the Apurinic-Apyrimidinic DNA Sites/Genotoxic Effects in Horse Blood Cells.

Isometamidium chloride (ISM) is a trypanocide for the prophylactic and therapeutic use against vector-borne animal trypanosomosis (mainly Surra caused by Trypanosoma evansi) and African animal trypanosomosis caused by T. congolense/T. vivax/T. brucei). ISM was found to be an efficient trypanocide for therapeutic/prophylactic use against trypanosomosis; however, it produces some local and systemic detrimental effects in animals. We synthesized isometamidium chloride-loaded alginate gum acacia nanoformulation (ISM SANPS) to lessen the detrimental side effects of isometamidium chloride (ISM) while treating trypanosomal diseases. We intended to determine the cytocompatibility/toxicity, and DNA deterioration/chromosomal structural or number changes (genotoxicity) of ISM SANPs using mammalian cells in a concentration-dependent manner. Apurinic/apyrimidinic (AP) sites are one of the major types of DNA lesions formed during base excision and repair of oxidized, deaminated, or alkylated bases. The intensity of the cellular AP site is an excellent marker of the deterioration of DNA quality. We thought it pertinent to quantify the AP sites in ISM SANPs-treated cells. Our investigations established a dose-dependent cyto-compatibility or toxicity and DNA impairment (genotoxicity) in ISM SANPs-treated horse peripheral blood mononuclear cells. ISM SANPs were biocompatible at various concentrations tested on the mammalian cells.

Multiplex PCR for rapid differential diagnosis of co-prevalent species of Theileria (Theileria annulata and Theileria orientalis) in cattle.

Theileriosis is a tick-borne disease that causes enormous losses in the dairy industry. There are several species of Theileria that can infect bovines. Generally, more than one species are prevalent in any geographical area; thus, chances of co-infections are high. Differentiation of these species may not be possible by microscopic examination or serological tests. Therefore, in this study, a multiplex PCR assay was standardized and evaluated for rapid and simultaneous differential detection of two species of Theileria viz., Theileria annulata and Theileria orientalis. Species-specific primers were designed to target the merozoite piroplasm surface antigen gene (TAMS1) of T. annulata and the major piroplasm surface protein gene of T. orientalis, yielding specific amplicon of 229 bp and 466 bp, respectively. The sensitivity of multiplex PCR was 102 and 103 copies for T. annulata and T. orientalis, respectively. The simplex and multiplex PCRs were specific and showed no cross-reactivity with other hemoprotozoa for either primer. For comparative evaluation, blood samples from 216 cattle were tested by simplex and multiplex PCR for both species. Using multiplex PCR, 131 animals were found infected for theileriosis, of which 112 were infected with T. annulata, five were infected with T. orientalis, and 14 had mixed infections. This is the first report of T. orientalis from Haryana, India. Representative sequences of T. annulata (ON248941) and T. orientalis (ON248942) were submitted in GenBank. The standardized multiplex PCR assay used in this study was specific, sensitive, for the screening of field samples.

Production of Bovine Rotavirus VP6 Subunit Vaccine in a Transgenic Fodder Crop, Egyptian Clover (Berseem, Trifolium alexandrinum) that Elicits Immune Responses in Rabbit.

Group A rotavirus causes acute gastroenteritis in young ones of animals worldwide and is responsible for a high rate of their morbidity and mortality leading to huge economic losses. Developing affordable and safer vaccine on large scale is imperative to reach cattle population worldwide for the long-term control of diarrhea. Rotavirus middle capsid protein layer, VP6, is the most immunogenic and highly conserved protein that induces immune responses against rotavirus. In the present study, bovine group A rotavirus VP6 protein has been expressed for the first time in a highly nutritious and palatable forage crop, Trifolium alexandrinum, using Agrobacterium tumefaciens-mediated stable nuclear transformation. Transgenic nature of the shoots regenerated from cotyledon explants and rooted on hygromycin-containing medium was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, reverse transcription-PCR (RT-PCR) and quantitative real-time PCR (qPCR), and protein expression and quantification by Western blot and enzyme-linked immune-sorbent assay (ELISA), respectively. The transformation efficiency of 2.10% was obtained. The highest amount of VP6 protein produced in a transgenic line was 402 ng/g fresh weights (0.03% of total soluble protein). Oral feeding of transgenic leafy shoots expressing VP6 protein stimulated systemic immunity by inducing significantly higher titers of anti-VP6 serum IgG antibodies in rabbit to reduce rotavirus infection. These transgenic fodder plants offer safer vaccine produced on large scale at low cost with reduced regulatory issues to improve livestock's health and wealth. These plants would be used as alternative to the current live attenuated vaccines to protect young calves against rotavirus infection.

A novel approach for estimation of anti-FMDV protective immunity generated in multiple vaccinated field animals.

In this study, forty serum samples from field buffaloes vaccinated with inactivated foot-and-mouth disease (FMD) vaccine were collected. These animals were multiple vaccinated with the above vaccine during previous years. The study was conducted to determine the actual status of the protective antibodies generated after vaccination. Initially, the serum samples were tested by Liquid phase blocking ELISA (LPBE), and only samples with titer more than 1.4 in LPBE were chosen for further analysis. These samples were tested with an in-house Gold Nanoparticle-based test for detection of anti-FMDV structural protein antibodies, in which the antibodies were detected at 10-4 dilution; this was suggestive of strong antibody titer generated post-vaccination. To test the binding affinity of these antibodies with the antigen, an avidity ELISA was developed and outcomes were expressed in terms of avidity index (AI). It was found that the avidity was low in some of the animals even after multiple vaccinations. Therefore, multiple vaccinations and strong antibody titer generation may not be the actual indicator of the protective immune response generated. We conclude that avidity ELISA can be a better approach than LPBE to measure the level of protective antibodies generated post-vaccination. Keywords: avidity ELISA; foot-and-mouth disease; post-vaccination monitoring; herd immunity; PCP-FMD.

Metal/metal oxide nanoparticles: Toxicity concerns associated with their physical state and remediation for biomedical applications.

Metal/metal oxide nanoparticles show promise for various applications, including diagnosis, treatment, theranostics, sensors, cosmetics, etc. Their altered chemical, optical, magnetic, and structural properties have differential toxicity profiles. Depending upon their physical state, these NPs can also change their properties due to alteration in pH, interaction with proteins, lipids, blood cells, and genetic material. Metallic nanomaterials (comprised of a single metal element) tend to be relatively stable and do not readily undergo dissolution. Contrarily, metal oxide and metal alloy-based nanomaterials tend to exhibit a lower degree of stability and are more susceptible to dissolution and ion release when introduced to a biological milieu, leading to reactive oxygen species production and oxidative stress to cells. Since NPs have considerable mobility in various biological tissues, the investigation related to their adverse effects is a critical issue and required to be appropriately addressed before their biomedical applications. Short and long-term toxicity assessment of metal/metal oxide nanoparticles or their nano-formulations is of paramount importance to ensure the global biome's safety; otherwise, to face a fiasco. This article provides a comprehensive introspection regarding the effects of metal/metal oxides' physical state, their surface properties, the possible mechanism of actions along with the potential future strategy for remediation of their toxic effects.

The Importance of Nanomedicine in Prophylactic and Theranostic Intervention of Bacterial Zoonoses and Reverse Zoonoses in the Era of Microbial Resistance.

Emergence of multidrug resistance (MDR), extensively drug resistance (XDR) and pandrug resistance (PDR) strains of bacteria in communicable diseases of zoonotic and reverse zoonotic importance is the major hurdle of one health concept. Increasing level of resistance against antibiotics among bacterial population throughout the world, slow pace of new antibacterial drug discovery and enhanced pace of resistance development by pathogenic bacteria possess major challenges for human and animal health as well as life in future. Alternative management strategy in terms of improved prophylactic vaccine; early, easy and effective diagnostics and therapeutic drugs against those resistant bacteria is the need of the hour. In this context nanomedicine can fit into the multifaceted demands as an effective prophylactic and theranostic alternative to control the communicable diseases in a cost effective manner in the era of microbial resistance. The current review is focused towards delineating the application of nanomaterials as vaccine or drug delivery system, diagnostics and directly acting antimicrobial therapeutic agents in combating the important zoonotic and reverse zoonotic bacterial diseases in recent scenario along with their potential benefits, limitations and future prospects to formulate successful eradication strategies.

Herbal Medicine for Urinary Tract Infections with the Blazing Nanotechnology.

Medicinal plants have been an integral and essential part of human life since ancient times. These have shaped the cultures around the globe. From underlings to elderly persons, everyone has come across to use herbal medicine for minor infection to deadly diseases. A wholesome approach is needed to maximize the knowledge about traditional resources. Thus, combining it with the new advents of technology is miraculous. Urinary tract infections (UTIs) are among the prevalent infections in the world. Increasing multi-drug resistance among uropathogens is quite problematic. The burning field of nanotechnology offers an enormous help in revolutionizing the diagnosis and treatment of the disease. The nanoparticles and nanocarriers can increase the bioavailability and efficacy of phytoconstituents targeted against the uropathogens. The present review focuses on herbal medicine and nanomaterials like nanoparticles, nanocarriers, nanoantibiotics as potent anti-bacterial agents against urinary tract infections.

A Novel Amperometric Genosensor for Rapid Detection of Canine Parvovirus in Feces.

A highly sensitive novel amperometric genosensor has been developed for rapid detection of canine parvovirus (CPV) DNA in fecal swabs of naturally infected dogs. The genosensor is based on a single stranded 5°-thiolated (SH) DNA probe complementary to VP1/VP2 gene of canine parvo vaccine strain, immobilized covalently on a polycrystalline gold (Au) electrode. The genosensor has been characterized by scanning electron microscopy (SEM), Fourier Transform Infra-Red Spectroscopy (FTIR), cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectra (EIS). The ssDNA-SH/Au electrode was hybridized with single stranded target DNA (ss T-DNA) in the sample. This hybridization was detected by reduction in current, generated by interaction of methylene blue (MB) with free guanine of ssDNA. The current response of genosensor was determined by CV, DPV and EIS. The sensor detected single stranded genomic DNA (ss g-DNA) isolated from vaccine strain of CPV in the range, 1.0-12.0 ng/µl at 25 °C for 10 min. Subsequently, the genobiosensor was applied for detection of CPV viral DNA in fecal swabs of naturally infected dogs. The limit of detection (LOD) of the sensor was 1.0 ng/µl of fecal viral DNA. To the best of our knowledge, this is the first report on development and application of amperometric biosensor for rapid, sensitive, specific point of care detection of viral DNA of CPV in feces.

Organoid Technology: A Reliable Developmental Biology Tool for Organ-Specific Nanotoxicity Evaluation.

Engineered nanomaterials are bestowed with certain inherent physicochemical properties unlike their parent materials, rendering them suitable for the multifaceted needs of state-of-the-art biomedical, and pharmaceutical applications. The log-phase development of nano-science along with improved "bench to beside" conversion carries an enhanced probability of human exposure with numerous nanoparticles. Thus, toxicity assessment of these novel nanoscale materials holds a key to ensuring the safety aspects or else the global biome will certainly face a debacle. The toxicity may span from health hazards due to direct exposure to indirect means through food chain contamination or environmental pollution, even causing genotoxicity. Multiple ways of nanotoxicity evaluation include several in vitro and in vivo methods, with in vitro methods occupying the bulk of the "experimental space." The underlying reason may be multiple, but ethical constraints in in vivo animal experiments are a significant one. Two-dimensional (2D) monoculture is undoubtedly the most exploited in vitro method providing advantages in terms of cost-effectiveness, high throughput, and reproducibility. However, it often fails to mimic a tissue or organ which possesses a defined three-dimensional structure (3D) along with intercellular communication machinery. Instead, microtissues such as spheroids or organoids having a precise 3D architecture and proximate in vivo tissue-like behavior can provide a more realistic evaluation than 2D monocultures. Recent developments in microfluidics and bioreactor-based organoid synthesis have eased the difficulties to prosper nano-toxicological analysis in organoid models surpassing the obstacle of ethical issues. The present review will enlighten applications of organoids in nanotoxicological evaluation, their advantages, and prospects toward securing commonplace nano-interventions.

Topical delivery of clobetasol propionate loaded nanosponge hydrogel for effective treatment of psoriasis: Formulation, physicochemical characterization, antipsoriatic potential and biochemical estimation.

Clobetasol propionate (CP), a superpotent topical corticosteroid, holds great promise for psoriasis treatment. However, common side effects like skin atrophy, steroidal acne, hypopigmentation and allergic contact dermatitis associated with it, hamper its utility for topical application. Taking this into consideration, the current work was aimed to fabricate CP loaded cyclodextrin nanosponge (CDNS) based hydrogel, to alleviate the aforementioned side effects, while controlling drug release. Nanosponges were crafted employing ß-cyclodextrin (polymer) and diphenyl carbonate (cross linker) and evaluated appropriately. The selected formulation augmented 45 folds water solubility, with respect to pure CP. The formulation possessed entrapment efficiency (56.33 ± 0.94%), particle size (194.27 ± 49.24 nm) with polydispersitive index (0.498 ± 0.095), surface charge (-21.83 ± 0.95 mV) and drug release (86.25 ± 0.28%). Selected CP-CDNS were found crystalline and uniform in size. Further, in vitro cell viability analysis has been performed using THP1 cells to evaluate cytocompatibility of CP nanosponges. The chosen CP nanosponges were then embedded into Carbopol hydrogel, and characterized for rheological behaviour, spreadability, and texture profile. The developed nanoformulations were also assessed in vivo using mouse tail model. Histological and biochemical assessments have been conducted to explore their antipsoriatic activity via oxidative stress biomarkers. The degree of orthokeratosis was observed remarkably (p < 0.001) amplified by CP-CDNS14 hydrogel as compared to untreated group (control) and CP hydrogel. In addition, drug activity and change in epidermal thickness were found significant. Our findings altogether advocated the profound potential of prepared CP nanogel in the topical treatment of psoriasis, with improved patient compliance.

Bovine brucellosis - a comprehensive review.

Brucellosis is a zoonotic disease of great animal welfare and economic implications worldwide known since ancient times. The emergence of brucellosis in new areas as well as transmission of brucellosis from wild and domestic animals is of great significance in terms of new epidemiological dimensions. Brucellosis poses a major public health threat by the consumption of non-pasteurized milk and milk products produced by unhygienic dairy farms in endemic areas. Regular and meticulous surveillance is essentially required to determine the true picture of brucellosis especially in areas with continuous high prevalence. Additionally, international migration of humans, animals and trade of animal products has created a challenge for disease spread and diagnosis in non-endemic areas. Isolation and identification remain the gold standard test, which requires expertise. The advancement in diagnostic strategies coupled with screening of newly introduced animals is warranted to control the disease. Of note, the diagnostic value of miRNAs for appropriate detection of B. abortus infection has been shown. The most widely used vaccine strains to protect against Brucella infection and related abortions in cattle are strain 19 and RB51. Moreover, it is very important to note that no vaccine, which is highly protective, safe and effective is available either for bovines or human beings. Research results encourage the use of bacteriophage lysates in treatment of bovine brucellosis. One Health approach can aid in control of this disease, both in animals and man.

Single Cell Metabolomics: A Future Tool to Unmask Cellular Heterogeneity and Virus-Host Interaction in Context of Emerging Viral Diseases.

Viral emergence is an unpredictable but obvious event, particularly in the era of climate change and globalization. Efficient management of viral outbreaks depends on pre-existing knowledge and alertness. The potential hotspots of viral emergence often remain neglected and the information related to them is insufficient, particularly for emerging viruses. Viral replication and transmission rely upon usurping the host metabolic machineries. So altered host metabolic pathways can be exploited for containment of these viruses. Metabolomics provides the insight for tracing out such checkpoints. Consequently introspection of metabolic alteration at virus-host interface has evolved as prime area in current virology research. Chromatographic separation followed by mass spectrometry has been used as the predominant analytical platform in bulk of the analyses followed by nuclear magnetic resonance (NMR) and fluorescence based techniques. Although valuable information regarding viral replication and modulation of host metabolic pathways have been extracted but ambiguity often superseded the real events due to population effect over the infected cells. Exploration of cellular heterogeneity and differentiation of infected cells from the nearby healthy ones has become essential. Single cell metabolomics (SCM) emerges as necessity to explore such minute details. Mass spectrometry imaging (MSI) coupled with several soft ionization techniques such as electrospray ionization (ESI), laser ablation electrospray ionization (LAESI), matrix assisted laser desorption/ionization (MALDI), matrix-free laser desorption ionization (LDI) have evolved as the best suited platforms for SCM analyses. The potential of SCM has already been exploited to resolve several biological conundrums. Thus SCM is knocking at the door of virus-host interface.

Detection methods for influenza A H1N1 virus with special reference to biosensors: a review.

H1N1 (Swine flu) is caused by influenza A virus, which is a member of Orthomyxoviridae family. Transmission of H1N1 occurs from human to human through air or sometimes from pigs to humans. The influenza virus has different RNA segments, which can reassert to make new virus strain with the possibility to create an outbreak in unimmunized people. Gene reassortment is a process through which new strains are emerging in pigs, as it has specific receptors for both human influenza and avian influenza viruses. H1N1 binds specifically with an α-2,6 glycosidic bond, which is present in human respiratory tract cells as well as in pigs. Considering the fact of fast multiplication of viruses inside the living cells, rapid detection methods need an hour. Currently, WHO recommended methods for the detection of swine flu include real-time PCR in specific testing centres that take 3-4 h. More recently, a number of methods such as Antigen-Antibody or RT-LAMP and DNA biosensors have also been developed that are rapid and more sensitive. This review describes the various challenges in the diagnosis of H1N1, and merits and demerits of conventional vis-à-vis latest methods with special emphasis on biosensors.

Evaluation of Japanese encephalitis virus E and NS1 proteins immunogenicity using a recombinant Newcastle disease virus in mice.

Japanese encephalitis (JE) is the most important cause of acute encephalitis syndrome (AES). Japanese encephalitis virus (JEV), the prototype member of the JE serocomplex, belongs to the genus Flavivirus. The immunogenic proteins envelope (E) and non-structural protein 1 (NS1) of JEV are widely explored for the development of vaccines and diagnostics against JEV. However, there are underlying concerns such as the risk of reversion of live-attenuated vaccines to high virulence, the incomplete inactivation of pathogens in inactivated vaccines and partial vaccine coverage. Newcastle disease virus (NDV) is an efficient viral vaccine vector to express several human and animal immunogenic proteins. In the present study, we have developed a recombinant NDV (rNDV), individually expressing the E and NS1 proteins of JEV (rNDV-Ejev and rNDV-NS1jev). The recovered rNDV-Ejev and rNDV-NS1jev were characterized in 9-day-old SPF embryonated chicken eggs and in cell culture. The vaccination of rNDV-Ejev and rNDV-NS1jev showed effective immunity against JEV upon intranasal immunization in BALB/c mice. The rNDVs vaccination produced effective neutralization antibody titers against both NDV and JEV. The cytokine profiling of the vaccinated mice showed an effective Th1 and Th2 mediated immune response. The study also provided an insight that E, when used in combination with NS1 could reduce the efficacy of only E based immunization in mice. Our results suggested rNDV-Ejev to be a promising live viral vectored vaccine against JEV. This study implies an alternative and economical strategy for the development of a recombinant vaccine against JEV.

Antiinflammatory peptides: current knowledge and promising prospects.

BACKGROUND: Inflammation is part of the regular host reaction to injury or infection caused by toxic factors, pathogens, damaged cells, irritants, and allergens. Antiinflammatory peptides (AIPs) are present in all living organisms, and many peptides from herbal, mammalian, bacterial, and marine origins have been shown to have antimicrobial and/or antiinflammatory properties. METHODS: In this study, we investigated the effects of antiinflammatory peptides on inflammation, and highlighted the underlying mechanisms responsible for these effects. RESULTS: In multicellular organisms, including humans, AIPs constitute an essential part of their immune system. In addition, numerous natural and synthetic AIPs are effective immunomodulators and can interfere with signal transduction pathways involved in inflammatory cytokine expression. Among them, some peptides such as antiflammin, N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), and those derived from velvet antler proteins, bee venom, horse fly salivary gland, and bovine ß-casein have received considerable attention over the past few years. CONCLUSION: This article presents an overview on the major properties and mechanisms of action associated with AIPs as immunomodulatory, chemotactic, antioxidant, and antimicrobial agents. In addition, the results of various studies dealing with effects of AIPs on numerous classical models of inflammation are reviewed and discussed.

Advances in Designing and Developing Vaccines, Drugs and Therapeutic Approaches to Counter Human Papilloma Virus.

Human papillomavirus (HPV) is a viral infection with skin-to-skin based transmission mode. HPV annually caused over 500,000 cancer cases including cervical, anogenital and oropharyngeal cancer among others. HPV vaccination has become a public-health concern, worldwide, to prevent the cases of HPV infections including precancerous lesions, cervical cancers, and genital warts especially in adolescent female and male population by launching national programs with international alliances. Currently, available prophylactic and therapeutic vaccines are expensive to be used in developing countries for vaccination programs. The recent progress in immunotherapy, biotechnology, recombinant DNA technology and molecular biology along with alternative and complementary medicinal systems have paved novel ways and valuable opportunities to design and develop effective prophylactic and therapeutic vaccines, drugs and treatment approach to counter HPV effectively. Exploration and more researches on such advances could result in the gradual reduction in the incidences of HPV cases across the world. The present review presents a current global scenario and futuristic prospects of the advanced prophylactic and therapeutic approaches against HPV along with recent patents coverage of the progress and advances in drugs, vaccines and therapeutic regimens to effectively combat HPV infections and its cancerous conditions.

Development of a rapid test for detection of foot-and-mouth disease virus specific antibodies using gold nanoparticles.

Foot and mouth disease is an economically important transboundary disease of wildlife and cloven hoofed animals including ruminants. In the absence of vaccination, detection of antibodies against structural proteins (SPs) of foot-and-mouth disease virus is an indicator of infection. In the present study, a rapid dot blot assay using gold nanoparticlees was developed for the detection of antibodies against SPs of FMDV. Commercially available FMD vaccine was used as a source of FMD antigen. After the synthesis of gold nanoparticles (GNPs), the GNP-dot blot assay was tested and was found very sensitive, as the detection of antibody was up to 10-4 of serum dilution. The GNP-dot assay was found specific as it didn't give dot with normal horse sera, fetal bovine sera and neonatal bovine calf serum samples when tested at 10-3 working dilution. When 30 serum samples from post-vaccinated buffaloes were tested at dilution of 10-3, all the samples were found positive with the variable intensity of dot. The synthesized GNPs and conjugated GNPS with antibody were characterized for their absorption limit, for their stability and for their approximate size. These characterized conjugated and non-conjugated GNPs were also analyzed by Transmission electron microscopy and Scanning electron microscopy. The GNP dot blot assay developed in this work gave promising results using vaccine antigen and can form an important tool for rapid diagnosis of FMD in FMD free countries, zones free of FMD and during last stage of FMD eradication when FMD vaccination will be withdrawn.