The S1, N and M proteins, obtained from the nephropathogenic N1/62 strain of infectious bronchitis virus (IBV) by immunoaffinity purification with monoclonal antibodies, were used for immunization of chickens. For all three antigens multiple immunizations were necessary for induction of an antibody response. Protection of chickens vaccinated with the S1 glycoprotein against virulent challenge was demonstrated by the complete absence of virus in tracheas and kidneys of vaccinated chickens. Following four immunizations with the S1 glycoprotein 71% and 86% of chickens were protected at the level of tracheas and kidneys, respectively. Three immunizations with the S1 glycoprotein protected 70% and 10% of chickens at the level of kidney and trachea, respectively. Neither the N nor the M antigen induced protection to a virulent challenge with the nephropathogenic N1/62 strain of IBV after four immunizations. Virus neutralizing, haemagglutination inhibiting and ELISA antibodies were detected in chickens immunized with the S1 glycoprotein and inactivated N1/62 virus, however there was no correlation between the presence of any of these antibodies and protection.

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Ten monoclonal antibodies (MAbs) directed against three structural proteins of infectious bronchitis viruses (IBV), the peplomer (S), membrane (M) and nucleocapsid (N) proteins, were characterized and used to determine the antigenic relationship between Australian IBV strains. One MAb (MAb 5) was directed against an epitope on the S 1 subunit of the peplomer, another (MAb 2) against an epitope on the M glycoprotein and eight MAbs (MAbs 1, 7, 9, 16, 24, 26, 27 and 51) were directed against seven non-overlapping epitopes on the N protein. None of the MAbs neutralized infectivity or inhibited haemagglutination of the virus. Conservation of the nine epitopes detected by these MAbs was determined in 13 serotypes of Australian IBV strains. Only epitope 5 on the S1 subunit of the peplomer was conserved in all strains. Epitope 2 on the M protein showed a high degree of conservation although this epitope was absent from four strains. None of the eight epitopes on the N proteins was conserved in all IBV strains but four epitopes (1, 16, 24 and 27) showed a high degree of conservation. Epitope 9 on the N protein was present only in IBV strains of one serotype whereas epitope 7 on the N protein distinguished vaccine viruses of serotype B from other IBV strains. The presence or absence of nine epitopes on three structural proteins differentiated IBV strains into five antigenic groups.

This paper describes mapping of antigenic and host-protective epitopes of infectious bronchitis virus proteins by assessing the ability of defined peptide regions within the S1, S2 and N proteins to elicit humoral, cell-mediated and protective immune responses. Peptides corresponding to six regions in the S1 (Sp1-Sp6), one in the S2 (Sp7) and four in the N protein (Np1-Np4) were synthesized and coupled to either diphtheria toxoid (dt) or biotin (bt). Bt-peptides were used to assess if selected regions were antigenic and contained B- or T-cell epitopes and dt-peptides if regions induced an antibody response and protection against virulent challenge. All S1 and S2 peptides were antigenic, being recognised by IBV immune sera and also induced an antibody response following inoculation into chicks. Three S1-and one S2-bt peptides also induced a delayed type hypersensitivity response indicating the presence of T-cell epitopes. The S2 peptide Sp7 (amino acid position 566-584) previously identified as an immundominant region, was the most antigenic of all peptides used in this study. Two S1 (Sp4 and Sp6) and one S2 peptide (Sp7), protected kidney tissue against virulent challenge. From four N peptides located in the amino-terminal part of the N protein, only one, Np2 (amino acid position 72-86), was antigenic and also induced a delayed type hypersensitivity response. None of the N peptides induced protection against virulent challenge. The results suggest that the S1 glycoprotein carries additional antigenic regions to those previously identified and that two regions located in the S1 and one in the S2 at amino acid positions 294-316 (Sp4), 532-537 (Sp6) and 566-584 (Sp7) may have a role in protection.

Australian infectious bronchitis viruses (IBV) have undergone a separate evolution due to geographic isolation. Consequently, changes occurring in Australian IBV illustrate, independently from other countries, types of variability that could occur in emerging IBV strains. Previously, we have identified two distinct genetic groups of IBV, designated subgroups 1 and 2. IBV strains of subgroup 1 have S1 and N proteins that share a high degree of amino acid identity, 81 to 98% in S1 and 91 to 99% in N. Subgroup 2 strains possess S1 and N proteins that share a low level of identity with subgroup 1 strains: 54 to 62% in S1 and 60 to 62% in N. This paper describes the isolation and characterisation of a third, previously undetected genetic group of IBV in Australia. The subgroup 3 strains, represented by isolate chicken/Australia/N2/04, had an S1 protein that shared a low level of identity with both subgroups 1 and 2: 61 to 63% and 56 to 59%, respectively. However, the N protein and the 3' untranslated region were similar to subgroup 1: 90 to 97% identical with the N protein of subgroup 1 strains. This N4/02 subgroup 3 of IBV is reminiscent of two other strains, D1466 and DE072, isolated in the Netherlands and in the USA, respectively. The emergence of the subgroup 3 viruses in Australia, as well as the emergence of subgroup 2 in 1988, could not be explained by any of the mechanisms that are currently considered to be involved in generation of IBV variants.

Sequencing of the S1 genes of nine Australian strains of infectious bronchitis virus (IBV) identified two genotypically distinct groups of strains. The strains Vic S, V5/90, N1/62, N3/62, N9/74 and N2/75 comprised group I, sharing 80.7-98-3 % identity in their deduced amino acid sequences. All group I strains were able to replicate in the trachea and kidney but only four strains, Vic S, N1/62, N9/74 and N2/75, were nephropathogenic, the latter three causing mortalities ranging from 32 to 96%. Group II contained strains N1/88, Q3/88 and V18/91 which only replicated in the trachea, inducing no mortalities. These viruses showed 72.3-92.8 % amino acid identity to each other and only 53.8-61.7 % identity to viruses of the first group. They were also distinct from the Massachusetts 41 and D1466 strains (47-5-55.7% amino acid identity). Thus N1/88, Q3/88 and V18/91 form a new group of viruses which are genotypically distinct from all previously characterized IBV strains. No definite correlations were established between the S1 amino acid sequences and the nephropathogenicity of strains.

Infectious bronchitis virus (IBV) is a coronavirus that causes upper respiratory, renal and/or reproductive diseases with high morbidity in poultry. Classification of IBV is important for implementation of vaccination strategies to control the disease in commercial poultry. Currently, the lengthy process of sequence analysis of the IBV S1 gene is considered the gold standard for IBV strain identification, with a high nucleotide identity (e.g. > or =95%) indicating related strains. However, this gene has a high propensity to mutate and/or undergo recombination, and alone it may not be reliable for strain identification. A real-time polymerase chain reaction (RT-PCR) combined with high-resolution melt (HRM) curve analysis was developed based on the 3'UTR of IBV for rapid detection and classification of IBV from commercial poultry. HRM curves generated from 230 to 435-bp PCR products of several IBV strains were subjected to further analysis using a mathematical model also developed during this study. It was shown that a combination of HRM curve analysis and the mathematical model could reliably group 189 out of 190 comparisons of pairs of IBV strains in accordance with their 3'UTR and S1 gene identities. The newly developed RT-PCR/HRM curve analysis model could detect and rapidly identify novel and vaccine-related IBV strains, as confirmed by S1 gene and 3'UTR nucleotide sequences. This model is a rapid, reliable, accurate and non-subjective system for detection of IBVs in poultry flocks.

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