Carnein, a serine protease from noxious plant weed Ipomoea carnea (morning glory)

A new serine protease from the latex of Ipomoea carnea spp. fistulosa (Morning glory), belonging to the Convolvulaceae family, was purified to homogeneity by ammonium sulfate fractionation followed by cation exchange chromatography. The enzyme, named carnein, has a molecular mass of 80.24 kDa (matrix-assisted laser desorption/ionization time-of-flight) and an isoelectric point of pH 5.6. The pH and temperature optima for proteolytic activity were 6.5 and 65 degrees C, respectively. The extinction coefficient (epsilon2801%) of the enzyme was estimated as 37.12, and the protein molecule consists of 35 tryptophan, 76 tyrosine, and seven cysteine residues. The effect of several inhibitors such as iodoacetic acid, diisopropylfluorophosphate, phenyl-methanesulfonyl fluoride, chymostatin, soybean trypsin inhibitor, HgCl2, 3S-3-(N-{(S)-1-[N-(4-guanidinobutyl)carbamoyl]3-ethylbutyl}carbamoyl)oxirane-2-carboxylic acid, N-ethyl maleimide, ethylene glycol-bis(alpha-amino ethyl ether)tetraacetic acid, ethylenediamminetetraacetic acid, and o-phenonthroline indicates that carnein belongs to the family of serine proteases. The enzyme is not prone to autolysis even at very low concentrations. The N-terminal sequence of carnein (T-T-H-S-P-E-F-L-G-L-A-E-S-S-G-L-X-P-N-S) exhibited considerable similarity to those of other plant serine proteases; the highest similarity was with alnus AG12, one of the subtilase family endopepetidases.     

Detection of Babesia bigemina DNA in ticks by DNA hybridization using a nonradioactive probe generated by arbitrary PCR

The Dig-labeled probe specific to Babesia bigemina generated from monomorphic RAPD fragment of approximately 873 bp size amplified by a 10 mer CGGTGGCGAA, detected up to 100 ng of template DNA. This nonradioactive probe also detected B. bigemina in preparations of larval tick DNA from two of the five samples on dot-blot hybridization.     

Identification of T-helper and linear B epitope in the hypervariable region of nucleocapsid protein of PPRV and its use in the development of specific antibodies to detect viral antigen

Peste des petits ruminants is a highly contagious viral disease of small ruminants making its diagnosis difficult from the similar symptoms of Rinderpest. Computer based prediction algorithms was applied to identify antigenic determinants on the nucleocapsid (N) protein of PPRV. Specificity and antigenicity of each peptide was evaluated by solid phase ELISA. Six specific peptide sequences were evaluated in multiple antigenic peptide (MAP) form and immune response was evaluated by supplementing universal T-helper epitope human IL-1beta peptide (VQGEESNDK, amino acids 163-171). Out of the six peptides 19mer sequence corresponding to 454-472 region of N protein of PPRV was found to be highly immunogenic and specific to PPRV. Evaluation of overlapping peptides differing in length for this 452-472 region, showed minimum length of 14 amino acid residues were required for the stable affinity binding of antigen-antibody. The results of immunization and indirect ELISA indicated the presence of T-helper epitope at the N-terminal end and linear B epitope at the C-terminal region of 454-472 19mer of nucleocapsid peptide of PPRV-nucleocapsid protein. The antipeptide antibodies developed against this region showed specificity to PPRV antigen differentiating it from RPV when used in indirect ELISA and western blot analysis.     

The plasmid constructs producing shRNA corresponding to the conserved 3D polymerase of Foot and Mouth Disease virus protects guinea pigs against challenge virus

RNA interference (RNAi) has been used as an effective antiviral strategy for its specific silencing of viral gene expression in mammalian cells. In this study, shRNA targeting two regions of Foot and Mouth Disease Virus (FMDV) i.e. 3D and 5'UTR which are very essential in virus replication were evaluated. The constructs were made using h7K RNA polymerase III promoter. We investigated in vivo inhibitory effect of shRNA on FMDV replication in BHK-21 cells and guinea pigs. The results showed that transfection of 3D shRNA could reduce virus growth by three folds when cells were challenged with 10(2) TCID(50) of FMDV. Pretreated guinea pigs with 3DshRNA were protected 80% with 10(3) GPID(50) of FMDV. As a first report in guinea pigs which are recognized animal model for FMD vaccine potency testing, the study suggests that shRNA could be a viable therapeutic approach to control severity of FMD infection and spread.     

Structure-function studies of Bubalus bubalis lingual antimicrobial peptide analogs

Antimicrobial peptides expressed on different epithelial lining are major components of the innate immune system. Based on the deduced amino acid sequence of Bubalus bubalis lingual antimicrobial peptide (LAP) cDNA (Accession No. DQ458768), five overlapping peptides LAP^23–55, LAP^42–64, LAP^21–64, LAP^1–26 and LAP^1–64 were synthesized using solid phase fluorenylmethoxycarbonyl (Fmoc) chemistry. Circular Dichroism spectroscopy of synthesized peptides revealed predominantly β-structure for LAP^23–55, LAP⁴²⁻⁶⁴ and LAP^21–64 with less α-helix in different solutions. Quantitation of secondary structure indicated the highest β-structure for all these three peptides in membrane mimetic SDS solution. The helicogenic solvent TFE could not induce helix in LAP^23–55 however TFE induced helical propensity was observed in LAP^42–64 and LAP^21–64. The quantitation of secondary structure indicated the highest ordered structure for LAP^23–55 followed by LAP^42–64 and LAP^21–64. The antibacterial activity of LAP^23–55 was found to be more potent against Staphylococcus aureus, Listeria monocytogens, Escherichia coli and Salmonella typhimurium followed by LAP^42–64 and LAP^21–64. Minimum inhibitory concentration (MIC) also showed similar trend with lowest value for LAP^23–55 followed by LAP^42–64 and LAP^21–64. Haemolysis and cytotoxicity was observed above 3 fold for LAP^21–64, above six fold for LAP^23–55 and LAP^42–64 of their MIC. The LAP^1–26 and LAP^1–64 could not produce any characteristic CD spectra and did not show any antimicrobial activity, indicating that N- terminal of the peptide negates the antimicrobial activity.     

Characterization of VP2 gene of an Indian Bluetongue virus serotype 2 and its close phylogenetic relationship to the Taiwan isolate

In this study we present the first report on partial amplification, sequencing and phylogenetic relationship of VP2 of the Indian isolate BTV-2. A PCR product of 1135 bp was amplified, cloned and sequenced. About 1063 bp of partial VP2 gene (1792-2854 bp region) of the Indian isolate was subjected to sequence analysis with already published sequences available in the genome database. The percent similarity of 85.2 was observed with Taiwan isolate and 59% with other isolates of BTV-2. However, 46.2% similarity with Australian BTV-1 and no significant similarity were noted with other serotypes. In-silico analysis and restriction enzyme digestion confirmed the presence of conserved SalI site at 2380 bp position in both Indian and Taiwan isolates. Phylogenetic analysis showed that all BTV-2 isolates formed one distinct group in which BTV-2 Indian and Taiwan isolate is more closely related and further demonstrated that BTV’s of the same serotype from different geographical regions were closely related at nucleotide and amino acid level, respectively.     

Identification of host plant resistance to pepper leaf curl virus in chilli (Capsicum species)

Three hundred and seven genotypes belonging to four cultivated and one wild species of Capsicum were screened against pepper leaf curl virus (PepLCV) causing devastating leaf curl disease of chilli (Capsicum annuum). Initial screening was done under field conditions based on coefficient of infection (CI), disease reaction to each genotype was assigned. Subsequently, selfed progenies of eight symptom-less and highly resistant lines were challenged by viruliferous white fly under glasshouse conditions, out of which only three genotypes, viz. GKC-29, BS-35 and EC-497636 showed no symptom. Using scion and root stalk of susceptible genotype (Pusa Jwala), these three putative symptom-less genotypes were further challenged by grafting and alternate grafting. The resistant reactions of GKC-29, BS-35, EC-497636 were confirmed because even after 50 days of successful grafting/alternate grafting, no viral symptom appeared on all the grafted plants of these genotypes. When subjected to PCR amplification with degenerate primers deigned to detect gemnivirus like PepLCV, the three symptom-less genotypes did not show any amplification, suggesting that the resistant reaction in three identified symptom-less resistant sources was because of the absence of viral genome and they are not symptom-less carrier.     

Changes in skin and rectal temperature in lactating buffaloes provided with showers and wallowing during hot-dry season

Twelve Murrah buffaloes in second or third parity during early lactation (50–70 days) were selected from the Institute’s herd. All the buffaloes were kept under loose housing system and were provided ad lib green maize fodder and water to drink during 30 days experiment during the month of August- September. The buffaloes were divided into two groups of six each. Showering group (SG) buffaloes were kept under water showers from 11:00 a.m. to 4:00 p.m., while wallowing group (WG) buffaloes were allowed to wallow in a water pond during the same time. Physiological responses viz. rectal temperature (RT), respiration rate (RR), pulse rate (PR) and skin temperature (ST) were recorded before (8.00 a.m.) and after (4.00 p.m.) showers or wallowing. Skin temperature at different sites i.e. trunk, forehead, udder, udder vein, and neck regions was measured. Skin and rectal temperature of both the groups were non significant in morning but varied (P < 0.01) in the evening. Skin temperature measured at all the sites was significantly lower (P < 0.01) in wallowing buffaloes than the showering group. Further, skin temperature of neck, head, udder, udder vein and RT varied (P < 0.01) in SG and WG buffaloes during periods of study. The significant changes in all the parameters of study further support the evidence on effective cooling of skin by wallowing in comparison to water showers. The correlation data indicated a positive correlation of maximum air temperature with RT in SG but correlation was non-significant in WG. RT was positively correlated with ST in SG (P < 0.05) and WG (P < 0.01). The pooled data analysis of both groups also indicated a positive correlation of maximum temperature with RT (P < 0.05). The morning respiration and pulse rate non-significantly varied in both group, however, in the evening, the respiration rate and pulse rate was more (P < 0.01) in SG in comparison to WG. No adverse effect of wallowing or shower treatment on mastitis incidence and general health of animals was observed.     

High-level expression of SAG1 and GRA7 gene of Toxoplasma gondii (Izatnagar isolate) and their application in serodiagnosis of goat toxoplasmosis

Toxoplasmosis, caused by Toxoplasma gondii, is a disease of economic importance in livestock, especially in sheep and goats, where it causes abortion. Although several serological tests are in use for diagnosis of infection, production of reliable reagents is a constraint. An 814 bp sequence coding for a truncated surface antigen surface antigen 1 (SAG1), a tachyzoite stage-specific protein, as well as a 657 bp sequence coding for granule protein 7 (GRA7), a dense granule protein were PCR amplified from the genomic DNA of T. gondii. The amplified products were ligated in pET-32b(+) and pET-32c(+) expression vectors, respectively and subsequently transformed into BL21(DE3)pLysS cells. A high-level expression of the histidine-tagged SAG1 and GRA7 fusion proteins were obtained after 7h of incubation. The recombinant proteins were purified using Ni-NTA column and were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis using reference positive sera from goat, rabbit and humans at 1:100 dilution. Subsequently, the diagnostic efficiency of the recombinant proteins, either individually or as a cocktail of the recombinant proteins, was assessed with 56 reference goat sera by enzyme-linked immunosorbent assay (ELISA). The immunoreactivity of the refolded SAG1 and GRA7 was evidenced by high OD values. The reactivity of the recombinant proteins as a cocktail preparation was more than that of individual proteins in ELISA and could detect accurately the infection in goats. This is the first report of serological detection of caprine toxoplasmosis by ELISA using a cocktail of recombinant Toxoplasma proteins.     

Evaluation of a monoclonal antibody-based approach for the selection of foot-and-mouth disease (FMD) vaccine strains

Foot-and-mouth disease (FMD) virus exists as seven serotypes within which are numerous variants necessitating careful selection of vaccine strains. Currently, a serological assay system based on the use of polyclonal vaccine antisera is widely used for this selection. However, inherent variability in the matching antisera used makes the tests poorly reproducible and difficult to interpret. In this study, we have explored the possibility of replacing or supplementing the polyclonal antibody (PAb)-based method with one based on use of monoclonal antibodies (MAb). Panels of MAbs raised against two serotype O vaccine strains were examined for reactivity with 22 field viruses, isolated over a 10-year period between 1991 and 2001. Antigenic site 2 was found to comprise more than one epitope. The sequence variation in capsid protein VP2 harbouring antigenic site 2 was analysed and the amino acid residues at positions 79 and 134 appeared to greatly influence the binding of site 2 MAbs. Prediction of antigenic match based on MAb reactivity did not correlate closely with the results of a PAb-based "gold-standard" method and it was concluded that a wider panel of MAbs are needed that recognise all protective epitopes present on the surface of FMD virus together with a better understanding of those epitopes which are important in conferring protection.