DEVELOPMENT OF NANOPARTICLES COUPLED INACTIVATED AND MULTI EPITOPE BASED RECOMBINANT INFECTIOUS LARYNGOTRACHEITIS VACCINE
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Date
2022
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Abstract
The focus of the present study was to isolate and characterize the infectious laryngotracheitis virus using ICP4 and TK genes and to develop chitosan and PLGA nanoparticles coupled inactivated ILT vaccine. Another focus was to design and express multi epitope based recombinant protein and to develop chitosan and PLGA nanoparticles coupled multiepitope recombinant vaccine and evaluating its immune responses in chickens.
A total of 27 out of 29 ILT suspected flocks were found to be positive by PCR using ICP4 and TK genes and were observed to have amplicon size of 635 and 649 bp respectively. The PCR positive tracheal samples were subjected to isolation of ILTV in 12 days old embryonated chicken eggs through chorioallantoic membrane route. Multiple pock lesions and thickening were noticed in the infected chorioallantoic membrane of embryonated chicken eggs after 120 hr of infection. Histopathological examination of infected CAM showed hemorrhage, congestion, epithelial hyperplasia with lymphocytic and heterophilic infiltration and the presence of syncytial cells with intranuclear inclusion bodies.
Out of 27 field isolates, the four field isolates of ILTV were sequenced for both ICP4 and TK genes. The ICP4 gene sequences of field ILTV isolates were observed to have identical nucleotide and amino acid sequences with CEO vaccine and vaccine-like isolates. All the four sequences had six nucleotide mutations at residues 3875 (T→C), 3927(C→T), 3951(T→C), 3982(G→A), 4017(G→A), and 4309(A→T) in comparison to TCO vaccine and vaccine-like strains. The ILTV sequences of this study had three unique amino acid mutations at residues 1292 (P→L), 1328 (G→R), and 1437 (T→S) in comparison to TCO vaccine and vaccine-like strains.
The TK gene of four field ILTV sequences was observed to have four nucleotide mutations at positions 441, 540, 594, and 755. The four ILTV sequences of TK gene had one unique amino acid mutation at residues 252. The amino acid threonine was found at position 252 in all the four TK gene sequences of this study as well as low virulent and vaccine reference strains but replaced with methionine in virulent reference strains.