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.

Six recently isolated field strains of infectious bursal disease virus (IBDV) were compared to vaccine strains at the antigenic and genetic level to ascertain the level of heterogeneity among Australian IBDV strains. Five strains, 01/94, 02/95, 03/95, 04/95 and 08/95, isolated at four locations in the state of Victoria, were antigenic variants. They failed to react with monoclonal antibodies directed against two different epitopes on the VP2 protein which were present in vaccine strains and one field isolate (06/95) from the state of New South Wales. Serum neutralization tests confirmed that these strains were antigenic variants as they were of a different subtype to that of vaccine strains. Sequence comparison of the hypervariable region of the VP2 proteins showed that the five Victorian strains had between 13 and 16 amino acid substitutions in comparison with vaccine strains. Four to six of these substitutions were in the two hydrophilic domains previously identified as being of importance in the formation of protective virus neutralizing antibodies. Comparison of these five variants to those isolated previously in the USA revealed little similarity at both the antigenic and genetic level. Phylogenetic analysis showed that Australian IBDV strains belong to a separate and distinct genetic group which is considerably heterogeneous. Overall the results indicate that the current Australian IBDV situation resembles that seen in the USA, with the existence of classical and variant IBDV strains, but neither the classical nor the variant strains found in Australia are closely related to those prevalent in the USA.

The nucleocapsid protein of coronaviruses has been considered highly conserved, showing greater than 94% conservation within strains of a given species. We determined the nucleotide sequence of the N gene and the 3' untranslated region (UTR) of eight naturally occurring strains of IBV which differed in pathogenicity and tissue tropism. In pairwise comparisons, the deduced amino acid sequences of N of five strains Vic S, N1/62, N9/74, N2/75, and V5/90 (group I) shared 92.3-98.8% identity. The three strains N1/88, Q3/88, and V18/91 (group II) shared 85.8-89.2% identity with each other, but only 60.0-63.3% identity with viruses of group I. Amino acid substitutions, deletions, and insertions occurred throughout the N protein and involved regions previously identified as being conserved. Despite the considerable variation observed between the two virus groups, all N proteins contained a high proportion of basic residues, 80% of which were conserved in position. In addition, all strains contained approximately 30 serine residues of which 10 were conserved, the majority occurring between positions 168 and 194. As for all other coronaviruses, the region between positions 92 and 103 was highly conserved. Hence, a large number of amino acid changes can be tolerated within the N protein without affecting its integrity or functioning. The 3' UTR immediately downstream from the N gene was highly heterogeneous with extensive deletions occurring in the group II strains.

Low pathogenicity avian influenza viruses (LPAIVs) are generally asymptomatic in their natural avian hosts. LPAIVs can evolve into highly pathogenic forms, which can affect avian and human populations with devastating consequences. The switch to highly pathogenic avian influenza virus (HPAIV) from LPAIV precursors requires the acquisition of multiple basic amino acids in the haemagglutinin cleavage site (HACS) motif. Through reverse genetics of an H5N1 HPAIV, and experimental infection of chickens, we determined that viruses containing five or more basic amino acids in the HACS motif were preferentially selected over those with three to four basic amino acids, leading to rapid replacement with virus types containing extended HACS motifs. Conversely, viruses harbouring low pathogenicity motifs containing two basic amino acids did not readily evolve to extended forms, suggesting that a single insertion of a basic amino acid into the cleavage site motif of low-pathogenic viruses may lead to escalating selection for extended motifs. Our results may explain why mid-length forms are rarely detected in nature. The stability of the short motif suggests that pathogenicity switching may require specific conditions of intense selection pressure (such as with high host density) to boost selection of the initial mid-length HACS forms.

Phage-displayed recombinant antibody libraries derived from splenic mRNA of chickens immunized with an Australian strain of infectious bursal disease virus (IBDV) were constructed as single chain variable fragments (scFv) by either overlap extension polymerase chain reaction (PCR) or sequential ligation of the individual heavy (V(H)) and light (V(L)) chain variable gene segments. Sequential cloning of the individual V(H) and V(L) genes into a newly constructed pCANTAB-link vector containing the synthetic linker sequence (Gly(4)Ser)(3) was more efficient than cloning by overlap extension PCR, increasing the library size 500 fold. Eighteen IBDV specific antibodies with unique scFv sequences were identified after panning the library against the immunizing antigen. Eight of the clones contained an identical V(H) gene but unique V(L) genes. In ELISA analysis using a panel of Australian and overseas IBDV strains, one scFv antibody was able to detect all strains, whilst 3 others could discriminate between Australian and overseas strains, classical and variant strains and Australian field strains and vaccine strains. In addition, some scFvs showed significant neutralization titres in vitro. This report shows that generation of chicken antibodies in vitro by recombinant means has considerable potential for producing antibodies of diverse specificity and neutralizing capacity.

An Indonesian very virulent (vv) strain of infectious bursal disease virus (IBDV), designated Tasik94, was characterised both in vivo and at the molecular level. Inoculation of Tasik94 into 5-week-old specific-pathogen-free (SPF) chickens resulted in 100% morbidity and 45% mortality. The complete nucleotide and predicted amino acid sequences of genomic segments A and B were determined. Across each of the three deduced open reading frames (ORFs), Tasik94 shared the greatest nucleotide homology to Dutch vv strain D6948. Phylogenetic analyses were performed using 15 full-length polyprotein sequences and a total of 105 VP2 hypervariable region sequences from geographically and pathogenically diverse strains. In each case, Tasik94 grouped closely with vv strains, particularly those from Europe. The deduced VP1, VP2, VP3, VP4 and VP5 protein sequences of Tasik94 were aligned with those from published strains and putative virulence determinants were identified in VP2, VP3 and VP4. Alignment of additional protein sequences across the VP2 hypervariable region confirmed that residues Ile[242], Ile[256] and Ile[294] were highly-conserved amongst vv strains, and may account for their enhanced virulence.