S. Sapats

Antigenic and sequence heterogeneity of infectious bursal disease virus strains isolated in Australia

Summary. 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.

Sequence analysis of the S1 glycoprotein of infectious bronchitis viruses: identification of a novel genotypic group in Australia

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.

Isolation of a variant infectious bronchitis virus in Australia that further illustrates diversity among emerging strains

Summary.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.

Identification of previously unknown antigenic epitopes on the S and N proteins of avian infectious bronchitis virus

Summary.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.

Characterisation of an Indonesian very virulent strain of infectious bursal disease virus

Summary.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.

Generation of chicken single chain antibody variable fragments (scFv) that differentiate and neutralize infectious bursal disease virus (IBDV)

Summary. 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 (VH) and light (VL) chain variable gene segments. Sequential cloning of the individual VH and VL genes into a newly constructed pCANTAB-link vector containing the synthetic linker sequence (Gly4Ser)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 VH gene but unique VL 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.

A long-term study of Australian infectious bronchitis viruses indicates a major antigenic change in recently isolated strains.

The antigenic relationship among 36 IBV strains isolated between 1961 and 1994 from vaccinated and non-vaccinated chicken flocks was determined. Based on the reaction with nine monoclonal antibodies (MAbs) in ELISA and polyclonal chicken sera in western blotting, IBV strains clearly fell into two distinct antigenic groups. Nineteen IBV strains isolated between 1961 and 1994 from various locations were antigenically related, having common cross-reactive epitopes on the peplomer S, the nucleocapsid N and the membrane M proteins. IBV strains within this classical group could be antigenically differentiated further by serotyping and by their reaction with MAbs. Seventeen IBV strains isolated between 1988 and 1994, shared only a minor degree of antigenic similarity with strains in the classical group. Strains in this novel group were antigenically related to each other and shared cross-reactive epitopes particularly on the N and M proteins. The novel IBV strains were not detected before 1988 and their origin is unknown. They appeared suddenly and almost simultaneously at two distant commercial sites, Redland Bay and Appin, and were also isolated at a third location in Victoria 3 years later. The Appin strains persisted on the site for 3 years without changes in antigenicity, including the serotype; however, following introduction of vaccination with novel strains a variant of new serotype was isolated. Variants isolated in Victoria on the other hand showed greater antigenic diversity and tendency for change. Novel strains have not displaced classical strains which continued to be isolated frequently.

Characterization of infectious bursal disease virus isolates from Indonesia indicates the existence ofvery virulent strains with unique genetic changes

Sequencing of the hypervariable region of viral protein VP2 of infectious bursal disease virus (IBDV) isolates obtained from non-vaccinated chickens in Indonesia showed that the majority (16/17) were closely related topublished very virulent (vv)IBDV strains. Four isolates contained identical amino acid sequences to Asian andEuropean wIBDVs, sharing w-specific amino acid residues 222(Ala), 256(Πe), and 294(Πe). Eight isolatesdiffered by one amino acid at position 222(Ala->Ser); however, this change did not alter the pathogenicity orantigenicity of these strains. Two isolates, with amino acid substitutions at positions 272(Π→Thr) and279(Asp→Asn), did not cause clinical disease or mortality, and were therefore considered to be naturallyoccurring, attenuated mutants of vvIBDV. The results illustrate variability that might occur among vvIBDVstrains.

Chicken recombinant antibodies specific for very virulent infectious bursal disease virus.

Summary.A phage-displayed single chain variable fragment (scFv) antibody library was constructed from the immune spleen cells of chickens immunized with very virulent infectious bursal disease virus (vvIBDV) strain CS89. A library consisting of around 9.2 × 107 clones was subjected to 3 rounds of panning against captured CS89 virus. Analysis of individual clones by nucleotide sequencing revealed at least 22 unique scFv antibodies binding to vvIBDV in ELISA. Testing of the scFv antibody panel in ELISA against classical, variant or vaccine strains and a wide variety of vvIBDV isolates from the UK, China, France, Belgium, Africa, Brazil, Indonesia and the Netherlands identified one antibody, termed chicken recombinant antibody 88 (CRAb 88) that was specific for vvIBDV. CRAb 88 was capable of recognizing all vvIBDV strains tested regardless of their country of origin and showed no reactivity with classical, variant or vaccine strains, lending support to the use of this scFv as a powerful diagnostic tool for the differentiation of vvIBDV strains. Immunoprecipitation studies revealed that CRAb 88 was directed towards a highly conformational epitope located within the major neutralizing protein VP2. Sequence analysis of the hypervariable region of VP2 of the IBDV strains tested indicate that Ile(256) and Ile(294) may play roles in binding of CRAb 88. This is the first reagent of its type capable of positively distinguishing vvIBDV from other IBDV strains.

Restriction fragment length polymorphism analysis of the VP2 gene of Australian strains of infectious bursal disease virus

Twenty-four Australian strains of infectious bursal disease virus (IBDV) were characterized by reverse transcription/polymerase chain reaction-restriction fragment length polymorphism and compared with previously published overseas strains. A primer pair designed to amplify a 743 base pair fragment of the VP2 gene was used and restriction fragment length polymorphism profiles were determined for each strain using three restriction enzymes, Bst NI, Mbo I and Ssp I. Australian strains comprised 12 molecular groups that were unique and distinct from overseas IBDV strains. A specific Ssp I site that is used to predict a very virulent IBDV phenotype was absent from all Australian strains, contrary to a previous finding by Jackwood and Sommer (1999). One Australian strain (N1/99) contained an Ssp I site; however, this was located at a different position than that found in very virulent IBDV strains. The results demonstrate that restriction fragment length polymorphism can be used to rapidly differentiate Australian IBDV strains from overseas strains. However, the existence of a large number of molecular groups might preclude its effectiveness for inter-strain differentiation.