Sequence analyses, hemagglutination activity and development of diagnostic assays of avian infectious bronchitis viruses
Date Issued
2010
Date
2010
Author(s)
Chen, Hui-Wen
Abstract
In the beginning one-third part of this dissertation (Unit 3 and 4), research has focused on the sequence analyses of IBVs isolates in Taiwan. The putative recombinant events of Taiwan IBVs isolated from 1992 to 2007 were investigated by phylogenetic analysis and simplot analysis. The 3’ 7.3 kb structural protein genome of eight Taiwan strains was directly sequenced. Frequent recombination events were identified among the Taiwan and China CK/CH/LDL/97I-type strains. Putative crossover sites were located in the S1, S2, 3b, M genes and the intergenic region between the M and 5a genes. All of the recombinants showed chimeric IBV genome arrangements originated from Taiwan and China-like parental strains. In addition, this study reports on a viral surveillance program in Taiwan from 2005 to 2006 with sampling conducted in poultry slaughter houses. Eight out of 47 flocks (17%) were IBV-infected, from which 13 IBV isolates were recovered. Eleven of 13 isolates (84.6%) clustered with Taiwan group I based on the S1 gene. One IBV isolate showed evidence of frequent recombination events with China-like IBVs in the S gene. Another isolate demonstrated the incorporation of China-like and H120-like genome fragments within the S2 gene and the M gene region, respectively. Some antigenic changes were found in the one-directional neutralization test. However, no positive selection pressures were related to those variations in the S1 genes among Taiwan IBVs. Field IBVs in Taiwan revealed intertypic genetic recombination and antigenic diversity.
For the second one-third part of this dissertation (Unit 5 and 6), molecular and serological diagnosis of IBV were the research topics. Since a heterologous Mass-serotype vaccine has been used in Taiwan for a decade, group-specific identification on virus and antibody has been a difficult problem. The Unit 5 reports on a rapid and reliable multiplex reverse transcriptase-polymerase chain reaction (mRT-PCR) assay for the genotyping of IBVs. Local IBV strains and commercially available vaccines were used for evaluating the viral genotyping assay. A number of field isolates and were examined for clinical application. The results showed that all of the examined IBVs were accurately genotyped by identifying the corresponding bands on agarose gels. The mRT-PCR assay was able to detect as low as 103, 105 and 103 viral RNA copies of the TW-I, TW-II and Mass-type strains, respectively. The mRT-PCR assay accurately detected and differentiated vaccine viruses from wild-type strains in the field. Another aspect, in order to understand the status of field infection, a monoclonal antibody (mAb) blocking ELISA (b-ELISA) against local IBVs was developed. The selected mAb showed specificity to Taiwan IBV strains but no cross reactivity against the vaccine strain H120. By using the hemagglutination inhibition (HI) test as a gold standard, the cut-off value, sensitivity and specificity of the b-ELISA were evaluated with 390 field samples. The type-specificity of detection was validated with a panel of chicken hyperimmune sera. The results showed that the b-ELISA demonstrated high sensitivity (97.96%) and specificity (97.16%) of detection. The agreement between the results of b-ELISA and HI test was statistically significant (Kappa = 0.95) and no significant difference between these two methods (McNemar p = 0.72). The b-ELISA specifically detected Taiwan IBV serotypes rather than other three IBV serotypes and sera against other avian pathogens. This b-ELISA provides type-specific antibody detection to the local IBV strains. It has the potential to serve as a rapid and reliable diagnostic method of IBV clinical infections in the field of Taiwan.
In the last one-third part of this dissertation (Unit 7 and 8), research started with the characterization of the hemagglutination activity (HA) in Taiwan IBV strains and ended up with discovering the cell-associated hemadsorption activity of baculovirus-derived S1 protein. The HA activity of 13 Taiwan IBV strains were investigated. The results showed 9 of 13 Taiwan IBV strains failed to show any HA activity after the neuraminidase treatment under standard or optimized protocols. This difference is not genotype-dependent. The amount of S1 gene did not equivalently correlate to the obtained HA titers. The presence of S1 protein in the prepared HA antigens was verified by western blot. The present study provides the information on the diversity of HA activity that Taiwan IBVs display and primary investigation on the factors that may influence the HA activity. Furthermore, to investigate the hemagglutination activity mediated by the S1 protein, the full S1 gene of the Taiwan IBV 2575/98 was cloned and expressed in Sf9 cells with the baculovirus expression vector system. Both of the recombinant S1 protein and the recombinant baculovirus possessed good reactivity with the chicken hyperimmune sera against several IBVs. Most notably, the baculovirus infected Sf9 cells acquired the ability to directly hemadsorb the chicken erythrocytes without neuraminidase treatment. The phenomenon of hemadsorption was inhibited by the IBV chicken antiserum, indicating the hemadsorption activity was specific induced by IBV-related proteins. However, no HA titer was obtained from any materials, even after additional neuraminidase treatment. This is the first observation of cell-associated hemadsorption activity in IBV research. This finding may provide a simple model for investigating the mechanism of virus-mediated hemagglutination activity, cell attachment and fusion.
Subjects
Blocking ELISA
Hemadsorption activity
Hemagglutination activity
Infectious bronchitis virus
Multiplex RT-PCR
Recombinant S1 protein
Sequence
SDGs
Type
thesis
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