Development of immunochromatographic strip assay to detect recent infection of Japanese encephalitis virus in swine population.

Japanese Encephalitis (JE) is a mosquito borne re-emerging viral zoonotic disease. Sero-conversion in swine occurs 2-3 weeks before human infection, thus swine act as a suitable sentinel for predicting JE outbreaks in humans. The present study was undertaken with the objective of developing immunochromatographic strip (ICS) assay to detect recent infection of Japanese Encephalitis virus (JEV) in swine population. The two formats of ICS assay were standardized. In the first format, gold nanoparticles (GNP) were conjugated with goat anti-pig IgM (50 µg/ml) followed by spotting of recombinant NS1 protein (1 mg/ml) of JEV on NCM as test line and protein G (1 mg/ml) as control line. In the format-II, GNP were conjugated with rNS1 protein (50 µg/ml) followed by spotting of Goat anti-pig IgM (1 mg/ml) as test line and IgG against rNS1 (1 mg/ml) as control line. To decrease the non- specific binding, blocking of serum and nitrocellulose membrane (NCM) was done using 5% SMP in PBS-T and 1% BSA, respectively. Best reaction conditions for the assay were observed when 10 µl of GNP conjugate and 50 µl of 1:10 SMP blocked sera was reacted on BSA blocked NCM followed by reaction time of 15 mins. Samples showing both test and control line were considered positive whereas samples showing only control line were considered negative. A total of 318 field swine sera samples were screened using indirect IgM ELISA and developed ICS assay. Relative diagnostic sensitivity and specificity of format-I was 81.25% and 93.0% whereas of format-II was 87.50% and 62.93%, respectively. Out of 318 samples tested, 32 were positive through IgM ELISA with sero-positivity of 10.06% while sero-positivity with format-I of ICS was 8.1%. Owing to optimal sensitivity and higher specificity of format-I, it was validated in three different labs and the kappa agreement ranged from 0.80 to 1, which signifies excellent repeatability of the developed assay to test field swine sera samples for detecting recent JEV infection.

An in silico approach for identification of lead compound as FtsZ inhibitor.

The remarkable conservation of the FtsZ among Gram-positive and Gram-negative bacteria, a crucial GTPase in bacterial cell division, has emerged as a promising antibacterial drug target to combat antibacterial resistance. There have been several coordinated efforts to develop inhibitors against FtsZ which can also serve as potential candidates for future antibiotics. In the present study, a natural product-like library (≈50,000 compounds) was employed to conduct HTVS against Staphylococcus aureus FtsZ protein (PDB Id: 6KVP). Additionally, molecular docking was carried out in two modes, SP and XP docking, using the Schrödinger suite. The glide scores of ligands obtained by XP docking were further summarized and compared with the control ligands (ZI1- co-crystal and PC190723-a compound undergoing clinical trial). Using the Prime-MM-GBSA approach, BFE calculations were performed on the top XP-scored ligands (≈598 compounds). These hits were also evaluated for ADMET parameters using the Qikprop algorithm, SwissADME, and in silico carcinogenicity testing using Carcinopred-El. Based on the results, ligand 4-FtsZ complex was considered for the 300 ns MDS analysis to get insights into its binding modes within the catalytic pocket of FtsZ protein. The analysis revealed that the amide linkage sandwiched between the triazole and 1-oxa-8-azaspirodecan-8-ium moiety (Val203) as well as the aminoethyl group present at 1st position on the triazole moiety (Leu209, Leu200, Asp210, and Ala202) were responsible for the FtsZ inhibitory activity, owing to their crucial interactions with key amino acid residues. Further, the complex also displayed good protein-ligand stability, ultimately predicting ligand 4 as a potent lead compound for the inhibition of FtsZ. Thus, our in silico findings will serve as a framework for in-depth in-vitro and in-vivo investigations encouraging the development of FtsZ inhibitors as a new generation of antibacterial agents.

Chemical composition and biological activities of Pittosporum eriocarpum Royle: an unexplored medicinal plant of Indian himalayan region.

In this study, different parts (leaf, bark, and fruit) of Pittosporum eriocarpum were investigated to explore its chemical composition and biological activities. The GC-MS analysis confirmed the presence of fifty-seven, eighty-one, and forty-six compounds in leaf, fruit, and bark extract, respectively. The important identified bioactive compounds include 1,3,4,5-tetrahydroxy-cyclohexanecarboxylic acid (quinic acid), falcarinol, tetradecanoic acid, and isopropyl myristate. Further, four polyphenolic compounds namely p-coumaric, chlorogenic, ferulic acid, and catechin were also identified and quantified in different parts through HPLC-PDA analysis. Of the studied parts of P. eriocapum, leaf extract contains the highest total phenolic, flavonoid, and tannin content, and exhibited potent antioxidant activity in ABTS assay. P. eriocarpum extracts also exhibited strong antimicrobial activity against gram-negative bacteria and showed considerable high protection against free radical-mediated DNA damage. To the best of our knowledge, this is the first detailed study of the chemical composition and biological activities of P. eriocarpum.

Polymorphism in the leucine-rich repeats of TLR7 in different breeds of chicken and in silico analysis of its effect on TLR7 structure and function.

Chicken toll-like receptor 7 (chTLR7) is a viral sensing pattern recognition receptor and detects ssRNA. The ligand binding site comprises leucine-rich repeats (LRRs) located in the ectodomain of chTLR7. Hence, any polymorphism in the binding site would modify its functional interaction with the ligand, resulting in varied strength of immune response. This study first aimed to compare the single nucleotide polymorphisms (SNPs) associated with the ligand binding site of TLR7 in three indigenous chicken breeds namely Aseel, Kadaknath, Nicobari along with an exotic breed White Leghorn. Four synonymous SNPs (P123P, I171I, N339N and L421L) and four non-synonymous SNPs (I121V, S135T, F356S and S447G) were identified among various breeds. We employed in silico tools to screen the pathogenic nsSNPs and one nsSNP was identified as having potential impact on chTLR7 protein. Moreover, sequence and structure-based methods were used to determine the effect of nsSNPs on protein stability. It revealed I121V, F356S, and S447G as decreasing the stability while S135T increasing the stability of chTLR7. Additionally, docking analysis confirmed that I121V and F356S reduced the binding affinity of ligands (R-848 and polyU) to chTLR7 protein. The results suggest that the nsSNPs found in this study could alter the ligand binding of chTLR7 and modify the immune response between different breeds further contributing to disease susceptibility or resistance. Further, in vitro and in vivo studies are needed to analyze the effect of these SNPs on susceptibility or resistance against various viral diseases in poultry.

Unravelling the role of long non-coding RNAs in modulating the Hedgehog pathway in cancer.

Cancer is a multifactorial pathological condition characterized by uncontrolled cellular proliferation, genomic instability, and evasion of regulatory mechanisms. It arises from the accumulation of genetic mutations confer selective growth advantages, leading to malignant transformation and tumor formation. The intricate interplay between LncRNAs and the Hedgehog pathway has emerged as a captivating frontier in cancer research. The Hedgehog pathway, known for its fundamental roles in embryonic development and tissue homeostasis, is frequently dysregulated in various cancers, contributing to aberrant cellular proliferation, survival, and differentiation. The Hh pathway is crucial in organizing growth and maturation processes in multicellular organisms. It plays a pivotal role in the initiation of tumors as well as in conferring resistance to conventional therapeutic approaches. The crosstalk among the Hh pathway and lncRNAs affects the expression of Hh signaling components through various transcriptional and post-transcriptional processes. Numerous pathogenic processes, including both non-malignant and malignant illnesses, have been identified to be induced by this interaction. The dysregulation of lncRNAs has been associated with the activation or inhibition of the Hh pathway, making it a potential therapeutic target against tumorigenesis. Insights into the functional significance of LncRNAs in Hedgehog pathway modulation provide promising avenues for diagnostic and therapeutic interventions. The dysregulation of LncRNAs in various cancer types underscores their potential as biomarkers for early detection and prognostication. Additionally, targeting LncRNAs associated with the Hedgehog pathway presents an innovative strategy for developing precision therapeutics to restore pathway homeostasis and impede cancer progression. This review aims to elucidate the complex regulatory network orchestrated by LncRNAs, unravelling their pivotal roles in modulating the Hedgehog pathway and influencing cancer progression.

Endophytic fungi from Himalayan silver birch as potential source of plant growth enhancement and secondary metabolite production.

Mountain biodiversity is under unparalleled pressure due to climate change, necessitating in-depth research on high-altitude plant's microbial associations which are crucial for plant survival under stress conditions. Realizing that high-altitude tree line species of Himalaya are completely unexplored with respect to the microbial association, the present study aimed to elucidate plant growth promoting and secondary metabolite producing potential of culturable endophytic fungi of Himalayan silver birch (Betula utilis D. Don). ITS region sequencing revealed that the fungal isolates belong to Penicillium species, Pezicula radicicola, and Paraconiothyrium archidendri. These endophytes were psychrotolerant in nature with the potential to produce extracellular lytic activities. The endophytes showed plant growth promoting (PGP) traits like phosphorus solubilization and production of siderophore, indole acetic acid (IAA), and ACC deaminase. The fungal extracts also exhibited antagonistic potential against bacterial pathogens. Furthermore, the fungal extracts were found to be a potential source of bioactive compounds including the host-specific compound-betulin. Inoculation with fungal suspension improved seed germination and biomass of soybean and maize crops under net house conditions. In vitro PGP traits of the endophytes, supported by net house experiments, indicated that fungal association may support the growth and survival of the host in extreme cold conditions.

MicroRNA-21's role in PTEN suppression and PI3K/AKT activation: Implications for cancer biology.

MicroRNA-21 (miR-21) was recognized as a key figure in the intricate web of tumor biology, with a prominent role in regulating the PTEN tumor suppressor gene and the PI3K/AKT cascade. This review elucidates the multifaceted interactions between miR-21, PTEN, and the PI3K/AKT signaling, shedding light on their profound implications in cancer initiation, progression, and therapeutic strategies. The core of this review delves into the mechanical intricacies of miR-21-mediated PTEN suppression and its consequent impact on PI3K/AKT pathway activation. It explores how miR-21, as an oncogenic miRNA, targets PTEN directly or indirectly, resulting in uncontrolled activation of PI3K/AKT, fostering cancerous cell survival, proliferation, and evasion of apoptosis. Furthermore, the abstract emphasizes the clinical relevance of these molecular interactions, discussing their implications in various cancer types, prognostic significance, and potential as therapeutic targets. The review provides insights into ongoing research efforts to develop miR-21 inhibitors and strategies to restore PTEN function, offering new avenues for cancer treatment. This article illuminates the critical function of miR-21 in PTEN suppression and PI3K/AKT activation, offering profound insights into its implications for cancer biology and the potential for targeted interventions.

Emerging roles of SnoRNAs in the pathogenesis and treatment of autoimmune disorders.

SnoRNAs (small non-coding RNAs) have recently gained prominence in autoimmune diseases, revealing their crucial role in modulating the immune response and contributing to disease pathogenesis. Initially known for their involvement in ribosomal RNA processing and modification, molecular biology and genomics advancements have uncovered their broader impact on cellular function, especially in autoimmune disorders. Autoimmune diseases represent conditions characterized by the immune system's erroneous attacks on self-tissues, encompassing disorders like systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis. The complex etiology of these conditions involves a delicate interplay of genetic and environmental factors. Emerging evidence suggests that snoRNAs initially recognized for their housekeeping roles, extend their influence on immune regulation through diverse mechanisms. SnoRNAs have been implicated in epigenetic modification, directly affecting the gene expression profiles of immune cells. Their ability to guide site-specific changes on ribosomal RNAs and other non-coding RNAs can significantly influence the translation of proteins involved in immune response pathways. Moreover, snoRNAs interact with key immune-related proteins, modulating their functions and subsequently impacting immune cell development, activation, and tolerance. Dysregulation of snoRNA expression has been observed in various autoimmune diseases, underscoring their potential as biomarkers for disease diagnosis, prognosis, and therapeutic targets. Manipulating snoRNA expression or activity is a promising therapeutic intervention avenue, offering the potential for personalized treatment strategies in autoimmune diseases. However, there remains a need for comprehensive research efforts to elucidate the precise molecular mechanisms underlying snoRNA-mediated immune modulation. Further investigations in this domain are essential to unravel the potential of snoRNAs in autoimmune disorders.

Intradermal Vaccination against Influenza with a STING-Targeted Nanoparticle Combination Adjuvant Induces Superior Cross-Protective Humoral Immunity in Swine Compared with Intranasal and Intramuscular Immunization.

The development of cross-protective vaccines against the zoonotic swine influenza A virus (swIAV), a potential pandemic-causing agent, continues to be an urgent global health concern. Commercially available vaccines provide suboptimal cross-protection against circulating subtypes of swIAV, which can lead to worldwide economic losses and poor zoonosis deterrence. The limited efficacy of current swIAV vaccines demands innovative strategies for the development of next-generation vaccines. Considering that intramuscular injection is the standard route of vaccine administration in both human and veterinary medicine, the exploration of alternative strategies, such as intradermal vaccination, presents a promising avenue for vaccinology. This investigation demonstrates the first evaluation of a direct comparison between a commercially available multivalent swIAV vaccine and monovalent whole inactivated H1N2 swine influenza vaccine, delivered by intradermal, intranasal, and intramuscular routes. The monovalent vaccines were adjuvanted with NanoST, a cationic phytoglycogen-based nanoparticle that is combined with the STING agonist ADU-S100. Upon heterologous challenge, intradermal vaccination generated a stronger cross-reactive nasal and serum antibody response in pigs compared with intranasal and intramuscular vaccination. Antibodies induced by intradermal immunization also had higher avidity compared with the other routes of vaccination. Bone marrow from intradermally and intramuscularly immunized pigs had both IgG and IgA virus-specific antibody-secreting cells. These studies reveal that NanoST is a promising adjuvant system for the intradermal administration of STING-targeted influenza vaccines.

New drug discovery of cardiac anti-arrhythmic drugs: insights in animal models.

Cardiac rhythm regulated by micro-macroscopic structures of heart. Pacemaker abnormalities or disruptions in electrical conduction, lead to arrhythmic disorders may be benign, typical, threatening, ultimately fatal, occurs in clinical practice, patients on digitalis, anaesthesia or acute myocardial infarction. Both traditional and genetic animal models are: In-vitro: Isolated ventricular Myocytes, Guinea pig papillary muscles, Patch-Clamp Experiments, Porcine Atrial Myocytes, Guinea pig ventricular myocytes, Guinea pig papillary muscle: action potential and refractory period, Langendorff technique, Arrhythmia by acetylcholine or potassium. Acquired arrhythmia disorders: Transverse Aortic Constriction, Myocardial Ischemia, Complete Heart Block and AV Node Ablation, Chronic Tachypacing, Inflammation, Metabolic and Drug-Induced Arrhythmia. In-Vivo: Chemically induced arrhythmia: Aconitine antagonism, Digoxin-induced arrhythmia, Strophanthin/ouabain-induced arrhythmia, Adrenaline-induced arrhythmia, and Calcium-induced arrhythmia. Electrically induced arrhythmia: Ventricular fibrillation electrical threshold, Arrhythmia through programmed electrical stimulation, sudden coronary death in dogs, Exercise ventricular fibrillation. Genetic Arrhythmia: Channelopathies, Calcium Release Deficiency Syndrome, Long QT Syndrome, Short QT Syndrome, Brugada Syndrome. Genetic with Structural Heart Disease: Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia, Dilated Cardiomyopathy, Hypertrophic Cardiomyopathy, Atrial Fibrillation, Sick Sinus Syndrome, Atrioventricular Block, Preexcitation Syndrome. Arrhythmia in Pluripotent Stem Cell Cardiomyocytes. Conclusion: Both traditional and genetic, experimental models of cardiac arrhythmias' characteristics and significance help in development of new antiarrhythmic drugs.

Unlocking new approaches to Urolithiasis management via Nutraceutical.

Urolithiasis, commonly known as kidney stones, is characterized by the formation of hard deposits in the urinary tract. These stones can cause severe pain and discomfort, and their management typically involves a combination of medical interventions and lifestyle modifications. According to the literature, 30% and 50% of urolithiasis cases recur. Between 9 and 12% of persons in industrialised countries are predicted to have urolithiasis at some time. Due to the high frequency of stone formation, recurrent nature, and prevalence in adults, it has a significant impact on society, the person, and the health care system. Adopting the best prophylactic measures is crucial in light of these developments to decrease the impact of urolithiasis on individuals and society. In recent years, there has been growing interest in the potential role of nutraceuticals in the management of urolithiasis. Nutraceuticals, such as herbal extracts, vitamins, minerals, and probiotics, have gained recognition for their potential in promoting urinary health and reducing the risk of urolithiasis. These compounds can aid in various ways, including inhibiting crystal formation, enhancing urine pH balance, reducing urinary calcium excretion, and supporting kidney function. Additionally, nutraceuticals can help alleviate symptoms associated with urolithiasis, such as pain and inflammation. While medical interventions remain crucial, incorporating nutraceuticals into a comprehensive management plan can offer a holistic approach to urolithiasis, improving patient outcomes and quality of life. Therefore, nutraceuticals may be a desirable choice for treating and avoiding recurring urolithiasis for patients and medical professionals. Therefore, the present study has focused on nutraceuticals' role in preventing urolithiasis.

Prognostic Potential of Thrombocyte Indices, Acute Phase Proteins, Electrolytes and Acid-Base Markers in Canine Parvovirus Infected Dogs With Systemic Inflammatory Response Syndrome.

Dogs with canine parvovirus enteritis (CPVE) that develop systemic inflammatory response syndrome (SIRS) frequently have a poor prognosis. The aim of the study was to assess the prognostic potential of thrombocyte indices, acute phase proteins, electrolytes, and acid-base markers in CPVE puppies with SIRS (CPVE-SIRS+) at admission. A case-controlled, prospective, and observational study was performed on 36 CPVE puppies. Mean concentrations of C-reactive protein (CRP), albumin, thrombocyte count, mean platelet volume (MPV), platelet distribution width (PDW), sodium (Na+), potassium (K+), chloride (Cl-) and ionized calcium (iCa) were measured and strong ion difference 3 (SID3), ATOT-albumin and ATOT-total protein were determined in CPVE-SIRS+ survivors and nonsurvivors. A prognostic cut-off value for predicting the disease outcome was determined by receiver operating characteristic (ROC) curve analysis. The mean values of MPV, PDW and CRP were significantly higher and the mean values of albumin, Cl- and ATOT-albumin were significantly lower in CPVE-SIRS+ nonsurvivor than CPVE-SIRS+ survivor puppies on the day of admission, but the thrombocyte count, Na+, K+, iCa, SID3 and ATOT- total protein values did not differ significantly. The positive predictive values (PPVs) for survival using cut-off value of MPV (≤15.08 fL), PDW (≤14.85%), CRP (≤180.7 mg/L), albumin (≥1.795 g/dL), Cl- (≥96.00 mmol/L), and ATOT-albumin (≥7.539) were determined as 100%, 100%, 100%, 80%, 100%, and 80%, respectively with better area under ROC curve and sensitivity. Based on sensitivity, specificity, and PPVs from ROC analysis, it is concluded that the determination of Cl- concentration and MPV at admission followed by CRP will serve as the most appropriate biomarkers in predicting the disease outcome of CPVE puppies that develop SIRS.

Antioxidant supplementation during treatment of outpatient dogs with parvovirus enteritis ameliorates oxidative stress and attenuates intestinal injury: A randomized controlled trial.

A prospective randomized controlled clinical study was conducted to determine whether antioxidant supplementation as an adjunct therapy alters hemogram, oxidative stress, serum intestinal fatty acid binding protein-2 (IFABP-2) level, fecal viral load, clinical score (CS) and survivability in outpatient canine parvovirus enteritis (CPVE) dogs. The dogs with CPVE were randomized to one of the five treatment groups: supportive treatment (ST) alone, ST with N-acetylcysteine (ST+NAC), resveratrol (ST+RES), coenzyme Q10 (ST+CoQ10) or ascorbic acid (ST+AA). The primary outcome measures were reduction of CS and fecal HA titre, and enhancement of survivability. Secondary outcome measures were reduction of oxidative stress indices and IFABP-2 level from day 0 to day 7. The mean CS and HA titre were significantly (P < 0.05) decreased from day 0 to 7 in ST and all antioxidant groups. The supplementations of NAC, RES and AA along with ST markedly (P < 0.05) reduced the concentrations of malondialdehyde, nitric oxide and IFABP-2 on day 7 as compared to ST alone. Additionally, NAC and RES supplementations markedly (P < 0.05) improved the total leukocyte count and neutrophil count in CPVE-affected dogs. NAC and RES could serve as better antioxidants for the amelioration of oxidative stress in CPVE but, the antioxidants did not confer any additional benefits in reduction of CS, fecal HA tire, or survivability when compared with ST alone.

The potentialities of omics resources for millet improvement.

Millets are nutrient-rich (nutri-rich) cereals with climate resilience attributes. However, its full productive potential is not realized due to the lack of a focused yield improvement approach, as evidenced by the available literature. Also, the lack of well-characterized genomic resources significantly limits millet improvement. But the recent availability of genomic data and advancement in omics tools has shown its enormous potential to enhance the efficiency and precision faced by conventional breeding in millet improvement. The development of high throughput genotyping platforms based on next-generation sequencing (NGS) has provided a low-cost method for genomic information, specifically for neglected nutri-rich cereals with the availability of a limited number of reference genome sequences. NGS has created new avenues for millet biotechnological interventions such as mutation-based study, GWAS, GS, and other omics technologies. The simultaneous discovery of high-throughput markers and multiplexed genotyping platform has aggressively aided marker-assisted breeding for millet improvement. Therefore, omics technology offers excellent opportunities to explore and combine useful variations for targeted traits that could impart high nutritional value to high-yielding cultivars under changing climatic conditions. In millet improvement, an in-depth account of NGS, integrating genomics data with different biotechnology tools, is reviewed in this context.

Effect of systemic inflammatory response syndrome on thrombocytogram, acute phase proteins, electrolytes, acid-base indices and cytokine expression in naturally canine parvovirus infected dogs.

Systemic inflammatory response syndrome (SIRS) in canine parvoviral enteritis (CPVE) is associated with high mortality in young puppies. Changes in acute phase response, thrombocytogram, inflammatory cytokine profiles, and disturbances in electrolyte and acid-base homeostasis are thought to have a significant impact on the development of SIRS. However, the mechanisms causing these perturbations have not been well described in CPVE puppies, especially with SIRS. The purpose of this study was to assess the changes of electrolytes, acid-base indices using strong ion model, acute phase proteins and thrombocytogram in blood and expressions of inflammatory cytokines in blood mononuclear cells of CPVE puppies with or without SIRS at admission. Additionally, the positive predictive value (PPV) and cut-off value with specificity and sensitivity of the biomarkers were determined by receiver operating characteristic (ROC) curve analysis to predict the development of SIRS in CPVE puppies at admission. A case-controlled, prospective and observational study was conducted on fifteen SIRS-positive CPVE, twenty-one SIRS-negative CPVE and six healthy puppies. Our data showed marked hyponatremia, hypokalemia, hypoalbuminemia and hypoproteinemia, decreased ATot-albumin and ATot-total protein and increased mean platelet volume (MPV), platelet distribution width (PDW) and C-reactive protein (CRP) concentration and up-regulation of TNF-α, IL-8 and IL-10 expressions in SIRS-positive CPVE puppies as compared to SIRS-negative CPVE puppies at admission. Based on sensitivity, specificity and AUC from ROC curve analysis and PPV, the CRP concentration in serum at a cut-off value of 141.9 mg/L and TLC of blood at a cut-off value of 3.355 × 103/µL were identified as potential prognostic biomarkers followed by ATot-total protein and total protein at a cut-off value of 11.80 and 4.72 g/dL, respectively to predict the development of SIRS in CPVE puppies at admission. In conclusion, the findings of the current study will help the canine practitioners to institute the time-sensitive and need based interventions to disrupt progression along the continuum of shock and multi-organ dysfunction syndrome in CPVE puppies that develop SIRS at admission.

LSDV126 gene based molecular assays for specific detection and characterization of emerging Lumpy Skin Disease virus.

Lumpy skin disease (LSD) is a highly infectious and economically important viral disease, which is currently emerging in the Indian subcontinent. LSD is caused by Lumpy Skin Disease Virus (LSDV) under the genus Capripoxvirus and the family Poxviridae. Since its first incursion in India in the year 2019, the virus is rapidly disseminating through different means like direct contact, fomites and mainly by blood-feeding insects. As the disease has never been reported from India or neighbouring countries, there is a lack of planning and preparatory measures in terms of diagnostics and vaccines to control the disease. In the absence of any homologous vaccine, a live attenuated heterologous goat pox vaccine (Uttarkashi strain) is now being widely used in the country for the prevention of LSDV infection. Use of live attenuated goat pox virus vaccine necessitates the availability of an assay which could specifically detect and differentiate LSDV from goat pox virus. In this study, nucleotide sequences of LSDV126 gene encoding extracellular enveloped virus protein of circulating LSDV and goat pox virus were determined and analyzed. Deletion of 27 nt tandem repeats was observed in LSDV in comparison to goat pox and LSDV vaccine viruses. The deletion region was targeted for designing primers specific to LSDV, but not goat pox virus. A novel isothermal polymerase spiral reaction (PSR) was optimized as pen side diagnostic for prompt and sensitive detection of genomic DNA of LSDV. The assay was found to be highly sensitive and specific when compared to the real-time PCR. The assay was found to be specifically detecting only LSDV but not the goat pox virus. The limit of detection was identified as 9 × 10-6 ng of positive DNA. The assay will provide a point of care tool that will be a boon for the successful control of LSD in India.

Evaluation of a Lipopolysaccharide and Resiquimod Combination as an Adjuvant with Inactivated Newcastle Disease Virus Vaccine in Chickens.

Various toll-like receptor (TLR) agonists have shown potential as adjuvants with different vaccines in both human and livestock species, including chickens. Our previous studies on combination of lipopolysaccharide (LPS; TLR4 agonist) and resiquimod (R-848; TLR7 agonist) showed the synergistic up-regulation of pro-inflammatory Th1 and Th2 cytokines in chicken peripheral blood mononuclear cells (PMBCs). Hence, the present study aimed to explore the combined adjuvant effect of LPS and R-848 with inactivated Newcastle disease virus (NDV) vaccine in chickens. Two weeks-old SPF chickens were immunized with inactivated NDV vaccine along with a combination of LPS and R-848 as an adjuvant with suitable control groups. A booster dose was given two weeks later. Antibody responses were assessed by enzyme linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test, while cell-mediated immune responses were analyzed by a lymphocyte transformation test (LTT) and flow cytometry following vaccination. Two weeks post-booster, the birds were challenged with a velogenic strain of NDV, and protection against clinical signs, mortality and virus shedding was analyzed. The results indicated that inactivated NDV vaccine with R-848 induced significantly higher humoral and cellular immune responses with 100% protection against mortality and viral shedding following a virulent NDV challenge. However, the combination of LPS and R-848 along with inactivated NDV vaccine produced poor humoral and cellular immune responses and could not afford protection against challenge infection and virus shedding when compared to the vaccine-alone group, indicating the deleterious effects of the combination on antigen-specific immune responses. In conclusion, the combination of LPS and R-848 showed the inhibitory effects on antigen-specific humoral, cellular and protective immune responses when used as an adjuvant with inactivated NDV vaccines in chickens. This inhibitory effect might have occurred due to systemic cytokine storm. A nanoparticle-based delivery of the combination of LPS and R-848 for slow and sustained release could be tried as an alternative method to explore the synergistic effect of the combination as an adjuvant in chickens.

Expression profiles of toll like receptors, MHC and cytokine genes along with viral load in organs of ducklings infected with an Indian isolate of duck enteritis virus.

A comprehensive study on the pathogenicity and host immune response was conducted in White Pekin ducklings after experimental infection with an Indian isolate of duck enteritis virus (DEV). The virus was found to be highly pathogenic and pantropic, which rapidly multiplied in various organs, mainly in the spleen and liver showing higher viral load with severe pathological lesions and caused 100% mortality. Expression profiles of immune gene transcripts in tissues (liver, spleen, brain) revealed upregulation of proinflammatory cytokines IFN-α, IFN- ß, IL-1ß, IL-6 and also iNOS with stimulation of TLRs (TLR-2, 3, 21). IFN-α was robustly upregulated (p < 0.05) especially in liver, might be playing role in antiviral innate immunity. Further, massive upregulation of MHC class-I (p < 0.01), expression of Th1 cytokines (IFN-γ & IL-2) and certain Th2 cytokines (IL-4 & IL-10) suggests stimulation of cell mediated as well as humoral immunity. To our knowledge, we are reporting first time about the robust upregulation of MHC class-I in spleen, liver and brain along with expression of certain cytokines in the peripheral blood mononuclear cells (PBMCs) during experimental DEV infection.

Regulatory T cells (Tregs) and their therapeutic potential against autoimmune disorders - Advances and challenges.

Autoimmune diseases are caused when immune cells act against self-protein. This biological self-non-self-discrimination phenomenon is controlled by a distinct group of lymphocytes known as regulatory T cells (Tregs), which are key inflammatory response regulators and play a pivotal role in immune tolerance and homeostasis. Treg-mediated robust immunosuppression provides self-tolerance and protection against autoimmune diseases. However, once this system fails to operate or poorly operate, it leads to an extreme situation where immune system reacts against self-antigens and destroys host organs, thus causing autoimmune diseases. Tregs can target both innate and adaptive immunity via modulating multiple immune cells such as neutrophils, monocytes, antigen-presenting cells, B cells, and T cells. This review highlights the Treg-mediated immunosuppression, role of several markers and their interplay during Treg development and differentiation, and advances in therapeutic aspects of Treg cells to reduce severity of autoimmunity-related conditions along with emphasizing limitations and challenges of their usages.

Development of IgM-ELISA for diagnosis of recent infection of Japanese encephalitis virus in equines.

Japanese encephalitis (JE) is a re-emerging mosquito borne disease, for which equines are most susceptible amongst all animals. Detection of specific immunoglobulin 'M' (IgM) is considered as an ideal way to diagnose recent JE virus infection in equines due to low virus load and short-term viremia. The present study was undertaken to develop a sensitive and specific recombinant NS1 protein based indirect IgM-ELISA and IgM capture (MAC) ELISA to diagnose recent infection of JEV in equines. Indirect IgM ELISA was standardized with relative diagnostic sensitivity and specificity of 100% and 88.5%, respectively. The validation of indirect IgM-ELISA in different laboratories revealed excellent reproducibility with Cohen's kappa value ranging between 0.84 and 1. The standardization of MAC ELISA was attempted using checker board titration method and non-specific binding of polyclonal anti-equine IgM capture antibody with anti-porcine IgG conjugate and with hyperimmune serum raised in swine against the antigen was observed. Hence, the MAC ELISA was standardized with monoclonal capture antibody; however, its diagnostic performance could not meet the satisfactory limit. Due to better sensitivity and less turnaround time, indirect IgM-ELISA was employed to screen 821 equine serum samples revealing 33.73% positivity of IgM antibodies against JEV in equine population of India. The high JEV sero-positivity warrants the need for vaccination in Indian equine population along with the demand for research focused towards anti-viral therapy. The indirect IgM-ELISA developed in the present study could be useful to diagnose acute or recent infection of JEV in equines as well as in sero-epidemiological studies.