Infectious Bronchitis Virus Diversity, Vaccine Alternatives, and Protective Immunity

Abstract

The work described in the current dissertation (i) examined the phenotypic effects of genetic changes in S1 genes of infectious bronchitis virus (IBV) isolates, assessed their pathogenicity in specific-pathogen-free (SPF) chickens, and tested the protective ability of the vaccine representing the parental virus against these isolates, (ii) evaluated the efficacy of a Newcastle disease virus-based recombinant IBV vaccine to replicate and induce mucosal and systemic antibody responses against NDV and IBV in commercial chickens with NDV and IBV maternal immunity, and (iii) characterized the humoral and cellular immune responses triggered by wild-type APMV-1 in chickens.

(i) Outbreaks of infectious bronchitis (IB) continue to occur, caused by novel variants of IB virus (IBV) emerging from selection of vaccine subpopulations and/or naturally occurring recombination events. Sequencing of spike subunit 1 (S1)-coding sequences of Arkansas (Ark)-type viruses obtained from clinical cases in Alabama broilers and backyard chickens shows both Ark Delmarva Poultry Industry (ArkDPI) vaccine subpopulations as well as apparent recombinants between Ark-type vaccine viruses and other IB vaccine viruses. IBV Ark-type isolates AL5, most similar to an ArkDPI vaccine subpopulation selected in chickens; AL4, showing a cluster of three nonsynonymous changes from ArkDPI subpopulations selected in chickens; and AL9, showing recombination with Massachusetts (Mass)-type IBV, were examined for pathogenicity and ability to break through immunity elicited by vaccination with a commercial ArkDPI vaccine. Analysis of predicted S1 protein structures indicated the changes were in regions previously shown to comprise neutralizing epitopes. Thus, they were expected to contribute to immune escape and possibly virulence. Based on clinical signs, viral load, and histopathology, all three isolates caused disease in naïve chickens, although AL9 and AL5 viral loads in trachea were statistically significantly higher (30- and 40-fold) than those of AL4. S1 gene sequencing confirmed the stability of the relevant changes in the inoculated viruses in the chickens, although virus in some individual chickens exhibited additional S1 changes. A single amino acid deletion in the S1 NTD was identified in some individual chickens. The location of this deletion in the predicted structure of S1 suggested the possibility that it was a compensatory change for the reduced ability of AL4 to replicate in the trachea of naïve chickens. Chickens vaccinated with a commercial ArkDPI vaccine at day of hatch and challenged at 21 days of age showed that vaccination provided incomplete protection against challenge with these viruses. Moreover, based on viral RNA copy numbers in trachea, differences were detected in the ability of the vaccine to protect against these IBV isolates, with the vaccine protecting the most poorly against AL4. These results provide additional evidence supporting that IBV attenuated vaccines, especially ArkDPI vaccines, contribute to perpetuating the problem of IB in commercial chickens.

(ii) Vaccination with a recombinant Newcastle disease virus (NDV) LaSota (LS) strain expressing Arkansas (Ark) -type infectious bronchitis virus (IBV) spike ectodomain (Se) protein and granulocyte macrophage colony-stimulating factor (GMCSF) (rLS/ArkSe.GMCSF) was evaluated in chickens of commercial origin with NDV and IBV maternally derived antibodies (MDA). Chickens were vaccinated ocularly with rLS/ArkSe.GMCSF at either 2, 8, 15 or 30 days of age. Control chickens were vaccinated with the rLS virus (not expressing IBV SE or GMCSF) on the same days. In addition, specific-pathogen-free (SPF) chickens were vaccinated with either virus at 2 days old. The results showed detection of NDV RNA in lacrimal fluids of vaccinated chickens, indicating successful replication of the recombinant virus at periocular mucosal sites. IBV IgA in lacrimal fluids and serum IBV antibodies were determined by ELISA using recombinant IBV-Ark-S1-protein-coated plates. Vaccination at 2 days of age with rLS/ArkSe.GMCSF in chickens with MDA elicited an IBV IgA response in lacrimal fluids. Chickens with MDA vaccinated with rLS/ArkSe.GMCSF at 8 days old showed IgA levels in lacrimal fluids not differing significantly from levels achieved after vaccination at 2 days of age. Vaccination at 30 days old did not result in increased IBV IgA levels in tear fluids of birds with MDA compared to unvaccinated birds with MDA. Vaccination with rLS/ArkSe.GMCSF of chickens with MDA resulted in limited IBV and NDV serum antibody responses. We conclude that vaccination with rLS/ArkSe.GMCSF induces IBV IgA at periocular mucosae, but limited serum antibody responses in chickens with NDV MDA.

(iii) The replication and immunogenicity of avian paramyxoviruses type 1 (APMV-1) Mottled duck/US(TX)/TX01-130/2001 and Mallard/US(MN)/MN00-39/2000 (designated WT-3 and WT-4, respectively) were evaluated in 14-day-old specific-pathogen-free (SPF) chickens. AMPV-1 WT-3 and WT-4 belong to class II genotype X. Immune responses were compared with responses elicited by the reference Newcastle disease virus (NDV) LaSota strain. Viral RNA was detected in half of lacrimal fluid samples from chickens inoculated with LaSota (6/12) or WT-4 (5/12) 8 days post-inoculation (DPI). Viral RNA was not detected in WT-3 inoculated chickens at 8, 14, or 21 DPI. These results suggest that the well-developed immune system of 14-day-old chickens was able to rapidly clear the APMVs. Seroconversion was observed in LaSota-, WT-3-, and WT-4-inoculated chickens. Similarly, increased levels of mucosal IgA detected in WT-3- and WT-4-inoculated chickens indicate that these wild-type APMV-1s triggered an early activation of mucosal immunity. By 14 DPI, LaSota induced the highest IgA levels, surpassing those elicited by both wild bird isolates and remaining elevated through 21 DPI. High antibody avidity was observed across all inoculated groups at 21 and 25 DPI with LaSota and WT-4 resulting in similar antibody avidity, while WT-3-inoculated chickens consistently displayed higher avidity antibodies. Although all three viruses elicited Bu1+ cell, cytotoxic T cell (CD3+CD8⁺) and helper T cell (CD3+CD4⁺) expansion in the Harderian gland (HG), differences were observed between them, with more robust cell-mediated responses being induced by WT-4. These results differ from other studies reporting similar expansion rates of T and B cells in HG by different NDVs, suggesting that strain-specific differences may influence mucosal immune responses. These findings contribute to our understanding of NDV immunology and highlight the importance of studying diverse viral isolates to develop vaccines to improve disease control.