Molecular Characterization of Infectious Bronchitis Virus Variants and Development of an Effective Vaccine

Abstract

The current study reports the seroprevalence of infectious bronchitis vIrus (my) in commercial eggs and meat-type chickens in Pakistan. The object ives of th e study were to monitor chicken flocks for the presence of various my strains, to determ ine their pathogenicity, to develop effective diagnostic measures for my strains and to develop and examine the efficacy of a killed my vaccine. Several commercial flocks (16 layers and 9 broilers) with a vaccination history against M-41 strain were bled and the serum samples were tested for the presence of antibodies against M-4I , 0 -274, 0 -1466 and 4-91 my strains. The strain M-41 was found to be most prevalent (100% in layers flocks and 77% in broiler flocks) followed by D-1466 (52%), D-274 (40%) and 4-91 (8%) was the lowest. The Haemagglutination Inhi bition (HI) titers were also determined and were found to be generally comparable between the layers and the broilers for a given [BY varian t. The IBY antigen from clinically [BY suspect chickens was also found to be detectable using the indirect immunofluorescence assay (IF A). Lungs and trachea were the only organs tested with IFA in which 40% of lungs showed positive for strain M-41 whereas only 10% of tracheas showed positive. The IBY M-41 strain was readily detectable in homogenate of these tissues. The direct haemagglutination assay (HA) was least sensitive however its sensitivity improved significantly when the homogenates were pretreated with phospholipase C (1 .3 % to 30.6%). In addition , agar gel precip itation test (AG PT) was also effe ct ive but detec ted only 5.3 % of the homogenate samples when tested for strain M-4l anti gen. Reverse Transcriptase Pol ymerase Chain Reaction (RT-PCR) was most sensitive and 57.3% of the tissue homogenate samples were found to be positi ve when tested agai nst S I OLIG05 ' and S 1 OLIG03 ' neucleotide primers. A total of 43 out of 75 IFA positive samples showed a viral PCR product around 1700 base pair. The ti ssue homogenates were passaged through the chicken embroynated eggs, wh ich sequentially increased their teratological effects with each passage. The effects included dwarfing, curling, stunting and urate deposits in challenged embryos. These effects were effectively neutralized by using mv variant specific antisera in a viral neutralization test. The mv variants isolated and present in the embryonic fluid were used to prepare a formalin-killed mv vaccine. This vaccine induced high levels of anti-mV titers as determined by haemagglutination inhibition assay. The booster vaccine inoculation enhanced the titers as expected. The vaccine protective effects could not be tested in this study. The results of this study which have shown the prevalence of various mv variants 111 chickens suggest that despite an existing vaccine program, mv infection is still prevalent in Pakistan. Nevertheless, by using proper diagnostic tools and reagents, the disease can be effectively and quickly diagnosed in affected flocks. In addition, indigenous vaccines representing the native mv strains would be most effective since mv is known to undergo significant antigenic changes overtime. Key words: Infectious Bronchitis Virus, lBV strains, Seromonitoring, HA, HI , AGPT, R T -PCR, teratological effects, vaccine.