Production of polyclonal viperin antisera using N-terminal deleted recombinant bovine viperin.

Viperin, also known as radical S-adenosyl methionine domain-containing protein (RSAD2) is a multifunctional interferon-stimulated gene (ISG) that is activated during the viral infections. Viperin belongs to S-adenosyl methionine (SAM) superfamily of enzymes known to catalyze radical-mediated reactions and viperin inhibits a wide range of DNA and RNA viruses through its broad range of activity. The present study reports cloning and expression of bovine viperin in a bacterial expression system. PCR-based site-directed mutagenesis was carried out for deletion of N-terminal 1-70 amino acid containing amphipathic helix of viperin that interferes in protein expression and purification. The resultant truncated viperin protein was expressed in Escherichia coli, BL-21(DE3) competent cells and purified using nickel charged affinity column. The truncated 54 kDa protein was confirmed by western blot using human RSAD2 as a probe. Further, in house, hyperimmune serum was raised against the truncated viperin in the rabbit and the reactivity was confirmed by western blot using mammalian expression vector construct of viperin transfected in Baby Hamster kidney (BHK) cells and in MDBK cells infected with Foot and Mouth disease Asia I virus.

Role of microRNA, bta-miR-375 in Immune Sturdiness of Vechur: The Native Cattle Breed of Kerala, India.

In the present study, next generation sequencing was employed to identify and explore the differential expression profiles of microRNAs (miRNAs) in peripheral blood mononuclear cells (PBMCs) of crossbred (B. taurus x B. indicus) and Vechur (B. indicus) cattle in response to the bacterial endotoxin-lipopolysaccharide (LPS). The PBMCs from adult apparently healthy female crossbred cows and Vechur cattle, a native cattle breed of Kerala, India were stimulated with 10 µg/mL of LPS for 6 h. Among the differentially expressed miRNAs, the expression of 13 miRNAs showed statistically significant up regulation while, significant decrease in the expression of 15 miRNAs was noticed in LPS treated PBMCs of Vechur cattle compared to crossbred cows. The expression profiling of miRNA, bta-miR-375, expression of which was found to be significantly down regulated in LPS treated PBMCs of Vechur cattle with respect to crossbred cattle by the NGS studies, is presented in the present manuscript. The decrease in expression of bta-miR-375 noticed by NGS was in accordance with the results of quantitative real time PCR assay. Functional gene enrichment analysis and pathway analysis revealed significant enrichment of predicted targets of bta-miR-375 in many immune related and cell signalling mechanisms. In addition, over representation of targets of bta-miR-375 was also noticed in pathogenesis of many of the bovine diseases. The study could also identify differences in the expression of cytokines, viz. Tumour Necrosis Factor Alpha (TNFα), Interleukin 4 (IL-4) and Interferon-γ (IFNγ) between LPS treated and untreated PBMCs of crossbred and Vechur cattle.

The Effect of Inorganic and Organic Zinc Supplementation on Growth Performance, Mineral Profile and Gene Expression Pattern of GLUT1 in Malabari Kids.

The objective of this experiment was to study and compare the effects of dietary supplementation of organic and inorganic zinc (Zn) on growth performance, nutrient utilisation and gene expression pattern of glucose transporter protein in peripheral blood mononuclear cells (PBMC) in Malabari kids. Fifteen, 3-4-month-old goat kids were divided into three groups uniformly by using completely randomised design (CRD). Group G1 was fed on basal diet as per NRC requirement, and G2 and G3 were fed on basal diet + 40 ppm Zn as inorganic zinc sulphate (ZnSO4) and 40 ppm Zn as organic Zn methionine, respectively, for a period of 91 days. Supplementation of inorganic and organic Zn had no significant effect on dry matter (DM) intake. The digestibility of crude protein (CP), ether extract (EE), neutral detergent fibre (NDF), hemicellulose and cellulose was significantly more in the organic Zn-supplemented group. The average daily gain and feed:gain ratio were significantly (p < 0.05) better in group G3 in comparison to G1 and G2, while the nitrogen retention was found to be (p < 0.01) higher in group G3 than in group G1. Zinc balance was found to be significantly (p < 0.01) increased in both supplemented groups with respect to unsupplemented group G1. The blood glucose level was (p < 0.01) lower in group G3 compared to group G1 suggesting the insulin-like activity of Zn. Serum Zn concentration was significantly (p < 0.01) increased in both Zn-supplemented groups. There was a significant (p < 0.05) rise in glucose transporter GLUT1 expression in groups G2 and G3 when compared to control group G1. Moreover, GLUT1 expression was found to be higher (p < 0.05) in group G3 as against the animals of group G2. Lowered blood glucose level might have stimulated more glucose transporter GLUT1 expression in PBMC. Organic Zn supplemented at 40 ppm level resulted in better growth performance, nutrient digestibility and nitrogen as well as Zn retention in goat kids. There was better absorption, and hence, less amount of Zn got excreted in the organic Zn-supplemented group.

Foot and mouth disease virus undergoes non-progressive replication in mice peritoneal macrophages and induces M1 polarization.

Despite the fact that macrophages link the innate and adaptive arms of immunity, it's role in the early infection of foot and mouth disease virus (FMDV) is largely unknown. Recently, depletion of macrophages in vivo after vaccination has shown to drastically diminish the protection against FMDV challenge in mouse model. Even the ability of macrophages to reduce or resist FMDV infection is not known hitherto. Therefore, we examined the replication ability of FMDV in mice peritoneal macrophages and the responsiveness in terms of macrophage polarization and cytokine production. Negative strand specific RT-PCR indicated replication of FMDV RNA in macrophages. Absolute quantitation of FMDV transcripts, immunofluorescence studies and titre of the infectious progeny virus revealed that replication peaked at 12 hpi and significantly declined by 18 hpi indicating non-progressive replication in the infected macrophages. Further, significant up regulation of inducible nitric oxide synthase by 8 -12 hpi and increase of M1 specific CD11c + cells by 42.6 % after infection showed that FMDV induce M1 polarization. A significant up regulation of TNFα and IL12 transcripts at 8 hpi supported that M1 macrophages were functional. Further, we studied the expression of Type I to III interferons (IFN) and other antiviral molecules. The results indicate a marked up regulation of Type I IFNα and ß by 9.2 and 11.2 fold, respectively at 8 hpi. Of the four IFN stimulated genes (ISG), viperin showed a significant up regulation by 286-fold at 12 hpi in the mice macrophages. In conclusion, the results suggest that replication of FMDV in mice peritoneal macrophages is non-progressive with up regulation of Type I IFN and ISGs. Further, FMDV induces M1 polarization in murine peritoneal macrophages.

Comparison of innate immune activation after prolonged feeding of milk fermented with three species of Lactobacilli.

The present investigation aimed at identifying the abilities of three different species of probiotic lactobacilli to modulate cellular immune responses in mouse neutrophils and macrophages in vivo over a study period of 60 days. Neutrophil respiratory burst enzymes (cytochrome c reductase and MPO) showed remarkable increased activity (P ≤ 0.01) after consumption of milks fermented by different species of probiotics over 30 and 60 days of feeding trials. Enzyme activities (ß-galactosidase and ß-glucuronidase) and nitric oxide production also increased considerably (P ≤ 0.01) in macrophages, both in peritoneal fluid and in enriched cell cultures. The effects of enhanced enzyme activities were corroborated by simultaneous increases in the phagocytic activities of neutrophils and macrophages. The increases in cellular functions were invariably maximal during the first 30 days of study and were maintained, but did not increase, over the next 30 days. Further, Lactobacillus helveticus-fed groups were most effective at modulating neutrophil functions whereas Lactobacillus paracasei-fed groups were more potent at enhancing macrophage functions. Together, our results indicate that probiotics have strain specific effects on stimulating cellular functions while not causing excessive stimulation of the immune system over longer feeding periods, thereby resulting in maximum and stable health benefits.