Rajesh ChandraPandey, Vinod Chandra2016-09-102016-09-102010-01http://krishikosh.egranth.ac.in/handle/1/76338Thesis-PhDRabies, a unique viral disease of warm blooded animals, kills virtually every individual it infects and is still a major problem for the developing and developed countries. Confirmatory diagnosis of rabies helps in taking appropriate prophylactic measures and is also essential for epidemiological investigations. The present study was undertaken with an aim to develop synthetic peptide antigens for serodiagnosis of rabies. At the same time antipeptide antibodies were developed for detecting rabies virus antigen. The rabies virus (CVS strain) was maintained in the laboratory by serial mouse to mouse passage. The rabies virus was also grown in BHK-21 and Neuro 2a cells. Additionally, the virus was also isolated from a cow calf suspected to have died of rabies virus infection. The Seller’s staining technique, fluorescent antibody test (FAT), rabies tissue culture infection test (RTCIT) and mouse inoculation tests were performed for confirmation of rabies virus. In case of Seller’s staining, magenta red colored Negri bodies were visible. In case of FAT and RTCIT, apple green colored, grain like fluorescent foci were observed in positive cases. Similarly, the intra-cerebral inoculation of mice with virus, characteristic symptoms were recorded and mice succumbs to death within seven days. The hyperimmune sera (HIS) was raised in mice and rabbits by immunizing with BPL inactivated CVS virus and also using commercially available ‘Raksharab’ vaccine. The virus titration was performed in suckling mice and BHK-21 cells to determine optimum concentration of virus for performing challenge studies. The LD50 of 10-6.5 and 10-3.9 were recorded for intra cerebral mouse inoculation and in cell culture. Thereafter, 20 to 50 LD50 dose of virus was used for various challenge experiments. For developing synthetic peptide antigens, the regions in Glycoprotein, Nucleoprotein and Phosphoprotein of rabies virus were identified based on parameters like hydrophylicity index, surface probability, antigenic index and presence of ordered Beta and turn structure. The identified peptides were synthesized in three different formats. Firstly, B- cell epitopes were synthesized as linear peptides. The chimeric linear peptides were synthesized by combining the B cell epitopes and the T- helper epitopes P & N. appropriate spacer was used between B cell and T helper epitope sequence. Besides this peptide sequences were also synthesized in multiple antigenic peptide (MAP) format in such a way that each MAP molecule contained 4 copies of peptide sequence. In total, 14 different peptides were synthesized by solid phase peptide synthesis method using Fmoc chemistry. The purity of peptides was checked by RP-HPLC and about 90% purity was recorded. For raising antipeptide antibodies, these 14 different peptides were injected in mice at 0,7,14, 28 days and sera were collected at 32 days. Besides, different combinations of these 12 peptides were also injected in mice to assess the role of T helper peptides in generating immune response in mice. The antigenic synthetic peptides detected the rabies virus specific antibodies in the HIS raised in both mice and rabbit. On repeated testing with peptide ELISA, at least 3 times more OD492 nm was recorded in comparison to preimmune sera. Additionally, when these peptides were reacted with convalescent serum from recovered mice, about 2 fold more increase in OD492 values were recorded in comparison to serum from infected mice. When peptides were used in combination to coat the ELISA plate, even better ELISA reactivity was observed, pointing towards suitability of these antigens in sero-diagnosis of rabies. The antipeptide antibodies were raised in mice using synthetic peptide antigen. The peptides were given individually and also in combination with T helper epitopes. Besides, the MAPs were also used for immunization either individually or in combinations with T helper epitope peptide. The antibody titer were estimated and it was found that when immunized along with T helper epitopes, the APA titer increased in case of linear epitopes. Whereas, in case of a combinations of MAPs & T helper epitopes , APA titers upto 102400 were also recorded. This shows the suitability of using T helper epitopes along with either linear or MAPs for generating high titer antibodies in the host. The APAs were also found suitable in detecting native virus by antigen capture ELISA. In double sandwich ELISA virus could be detected with 3 fold more ELISA signals than negative controls. The circular dichorism (CD) spectroscopy was performed to determine the solution conformation of linear, chimeric and MAPs. The CD spectra were recorded in 2 different solvents (water and 90% Tri fluoro ethanol). It was observed that peptides adopted ordered structure in both water and TFE with random coil structure ranging in between 20 to 35 %. When CD spectra of peptides were recorded in helicogenic solvent like TFE, the ordered structure, predominantly helix, was induced in most of the antigenic peptides at the expense of randomness. When peptides were coated on ELISA plate in 90% TFE, higher ELISA signals were recorded indicating that antigenic peptides in more ordered confirmation can serve as better ELISA antigens. Overall, in present study peptides were synthesized in different formats and their suitability in detecting antibodies was assessed. Further, the antipeptide antibodies raised against these peptide antigens have shown promising results for the diagnosis of native rabies virus. Basic virological techniques like virus isolation in cell culture and virus titration in vitro and in vivo were used to accomplish research objectives. The immunological techniques like FAT, RTCIT & ELISAs were also performed. Besides this, biophysical techniques like HPLC and CD spectroscopy were also performed to accomplish the desired objectives. The synthetic peptide antigens and epitope specific antipeptide antibodies developed in this study can be promising candidate reagents for the confirmatory diagnosis of rabies.enantigens, peptides, rabies, animal diseases, viral diseases, antibodies, diagnosisThesis