Development of recombinant capsid protein-based single serum dilution ELISA for sero-detection of porcine circovirus type 2 infection in pigs.

Porcine Circovirus 2 (PCV2) is the major causative agent of postweaning multisystemic wasting syndrome (PMWS) of swine and is one of the reasons for severe economic loss in swine industry. In India, there is a considerable prevalence rate of PCV2 infection in pig population, PCV2d being the most prominent genotype. Proper sero-diagnosis and sero-surveillance of the disease is formulated as an effective control measure. In this study, a recombinant capsid protein-based single serum dilution indirect ELISA was developed for determination of antibody titre of the infected pigs. The capsid protein (Cap) of PCV2d was produced in Saccharomyces cerevisiae cells and the capsid protein was purified by affinity chromatography. This recombinant protein was used as a coating antigen to develop a cost effective, highly sensitive and specific single serum dilution ELISA. The in-house developed ELISA was optimized to be used in a 1:200 single serum dilution. The developed ELISA along with a commercial ELISA kit were compared with a sensitive immuno-peroxidase assay (IPMA) by receiver-operating characteristics (ROC) test. Our results showed that the developed single serum dilution ELISA had a higher sensitivity and specificity in comparison to the commercial ELISA. The area under the ROC curve (AUC) also confirmed that the developed ELISA had a better overall diagnostic performance in comparison to the commercial ELISA kit.

Newcastle Disease Virus Vectored Chicken Infectious Anaemia Vaccine Induces Robust Immune Response in Chickens.

Newcastle disease virus (NDV) strain R2B, with an altered fusion protein cleavage site, was used as a viral vector to deliver the immunogenic genes VP2 and VP1 of chicken infectious anaemia virus (CIAV) to generate a bivalent vaccine candidate against these diseases in chickens. The immunogenic genes of CIAV were expressed as a single transcriptional unit from the NDV backbone and the two CIA viral proteins were obtained as separate entities using a self-cleaving foot-and-mouth disease virus 2A protease sequence between them. The recombinant virus (rR2B-FPCS-CAV) had similar growth kinetics as that of the parent recombinant virus (rR2B-FPCS) in vitro with similar pathogenicity characteristics. The bivalent vaccine candidate when given in specific pathogen-free chickens as primary and booster doses was able to elicit robust humoral and cell-mediated immune (CMI) responses obtained in a vaccination study that was conducted over a period of 15 weeks. In an NDV and CIAV ELISA trial, there was a significant difference in the titres of antibody between vaccinated and control groups which showed slight reduction in antibody titre by 56 days of age. Hence, a second booster was administered and the antibody titres were maintained until 84 days of age. Similar trends were noticed in CMI response carried out by lymphocyte transformation test, CD4+ and CD8+ response by flow cytometry analysis and response of real time PCR analysis of cytokine genes. Birds were challenged with virulent NDV and CIAV at 84 days and there was significant reduction in the NDV shed on the 2nd and 4th days post challenge in vaccinated birds as compared to unvaccinated controls. Haematological parameters comprising PCV, TLC, PLC and PHC were estimated in birds that were challenged with CIAV that indicated a significant reduction in the blood parameters of controls. Our findings support the development and assessment of a bivalent vaccine candidate against NDV and CIAV in chickens.

In-silico designing of epitope-based vaccine against the seven banded grouper nervous necrosis virus affecting fish species.

Neural necrosis virus (NNV) of family Nodaviridae affect wide range of fish species with viral encephalopathy and retinopathy causing mass mortality up to 100%. Currently there is no effective treatment and depopulation is only suggested recommendation. New avenues and approach are required to control this harmful malady. In this study we developed an epitope-based vaccine (EBV), against NNV using computation approach. We have selected two conserved proteins RNA-dependent RNA polymerase (RdRP) and capsid proteins. Based on more than ~ 1000 epitopes we selected six antigenic epitopes. These were conjugated to adjuvant and linker peptides to generate a full-length vaccine candidate. Biochemical structural properties were analyzed by Phyre2 server. ProtParam, Molprobity. Ramachandran plot results indicate that 98.7% residues are in a favorable region and 93.4% residues in the favored region. The engineered EBV binds to toll like receptor-5 (TLR5) an important elicitor of immune response. Further molecular docking by PatchDock server reveals the atomic contact energy (i.e. - 267.08) for the best docked model of EBV and TLR5 receptor. The molecular simulation results suggest a stable interaction; the RMSD and RMSF values are 1-4 Ǻ and 1-12Ǻ, respectively. Further we have suggested the best possible codon optimized sequence for its cloning and subsequent purification of the protein. Overall, this is a first report to suggest an in-silico method for generation of an EBV candidate against NNV. We surmise that the method and approach suggested could be used as a promising cure for NNVs.

Evaluation of a fusion gene-based DNA prime-protein boost vaccination strategy against Newcastle disease virus.

The low potency of genetic immunization has to date impeded development of commercial vaccines against major infectious diseases. The aim of this study was to develop and evaluate a fusion gene-based DNA prime-protein boost vaccination strategy to improve the efficacy of both DNA and subunit vaccines against Newcastle disease virus (NDV). The fusion (F) protein, a viral surface glycoprotein, is responsible for the cell membrane fusion and spread, also is one of the major targets for immune response. In this study, groups of chickens were vaccinated twice intramuscularly at 14-day interval either with plasmid DNA encoding F protein gene of NDV or with recombinant F protein alone or with plasmid DNA and boosted with the recombinant F protein and compared with birds that were vaccinated with live NDV vaccine. The immune response was evaluated by indirect ELISA, lymphocyte transformation test, virus neutralization test, cytokine analysis, immunophenotyping of peripheral blood mononuclear cells, and protective efficacy study against virulent NDV challenge virus infection. Chickens in prime-boost group developed a higher level of humoral and cellular immune responses as compared with those immunized with plasmid or protein alone. The DNA prime-protein boost using F protein of NDV yielded 91.6% protection against virulent NDV challenge infection better than immunization with DNA vaccine (66.6%) or rF protein (83.3%) alone. These findings suggest that the "DNA prime-protein boost" approach using full-length F gene could enhance the immune response against NDV in the chickens.

Development and evaluation of a Salmonella typhimurium flagellin based chimeric DNA vaccine against infectious bursal disease of poultry.

Infectious bursal disease (IBD) is an acute immunosuppressive disease of young chicks, caused by a double-stranded RNA virus. VP2 being the major capsid protein of the virus is an ideal vaccine candidate possessing the neutralizing epitopes. The present study involves the use of flagellin (fliC) as a genetic adjuvant to improve the immune response of VP2 based DNA vaccine against IBD. Our findings revealed that birds immunized with plasmid pCIVP2fliC showed robust immune response than pCIVP2 immunized groups. Further, challenge study proved that genetic fusion of fliC and VP2 can provide a comparatively higher level of protection against vvIBDV challenge in chickens than VP2 alone. These results thus indicate that Salmonella flagellin could enhance the immune responses and protection efficacy of a DNA vaccine candidate against IBDV infection in chickens, highlighting the potential of flagellin as a genetic adjuvant in the prevention of vvIBDV infection.

Analysis of Gill Structure from a Fresh Water Fish (Heteropneustes fossilis) Exposed to Bleached Sulfite Pulp Mill Effluents.

The present communication reports toxic effects of bleached sulfite pulp mill effluents in fish (Heteropneustes fossilis) gills, with optical, scanning electron, and transmission electron microscopy. The general adverse effects include dilation of the primary lamellar wall, curling of secondary lamellar terminals, displacement of epithelial cell layers, degeneration of secondary lamella, deposition of mucous, and severe congestion in the gill arch. The significant shortening of secondary lamellae, widening of lamellar tips, and significant decrease in the number of mitochondria in chloride cells as compared to controls are some specific effects of bleached sulfite pulp mill effluents. Scanning electron microscopy demonstrated tearing of tissues in gill lamellae and arches. Transmission electron microscopy revealed membrane distortion of mitochondria in chloride cells, loss of uniformity of microvilli in pavement cells, and abnormalities in nuclear shape in different cells of effluent-exposed fish gills. Toxicity of the bleached sulfite pulp mill effluents and its impact on fish are discussed in the light of existing literature. Further, the importance of microscopy in toxicological evaluation of environmental pollutants is emphasized in view of its specific application potential.