Jagoda Ignjatovic

The S1 glycoprotein but not the N or M proteins of avian infectious bronchitis virus induces protection in vaccinated chickens

SummaryThe 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.

A recombinant fowl adenovirus expressing the S1 gene of infectious bronchitis virus protects against challenge with infectious bronchitis virus

The spike peplomer S1 subunit sequence from avian infectious bronchitis virus (IBV) Vic S strain was expressed in a plasmid under the control of the fowl adenovirus (FAV) major late promoter (MLP). Two recombinants were constructed in FAV serotype 8 (FAV 8) by inserting the expression cassette between the SnaBI and XbaI restriction enzyme sites (clone DA3) or between the SpeI sites (clone CA6-20). Expression of the S1 gene in the recombinants was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) by 20h post-infection. Commercial broiler chickens were orally vaccinated at day 0 or day 6 post-hatch and challenged at day 35 post-hatch. FAV antibody ELISA confirmed that maternal antibody directed against inclusion body hepatitis (serotype 8) had decayed in control birds and that FAV specific serum IgG responses were produced in vaccinated birds at the time of challenge. Further, an S1 specific antibody response was detected prior to challenge. Birds were challenged with either Vic S (serotype B) or N1/62 (serotype C) strains of IBV. The tracheas of challenged birds were analyzed by RT-PCR and re-isolation of virus. In birds vaccinated at day 6, 90-100% protection at the trachea was induced against either homologous or heterologous challenge. The construction of a recombinant FAV expressing S1 of IBV demonstrates the potential of an alternative vaccination strategy against IBV.

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.

Rapid detection and non-subjective characterisation of infectious bronchitis virus isolates using high-resolution melt curve analysis and a mathematical model

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. ≥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.

Monoclonal antibodies to three structural proteins of avian infectious bronchitis virus: characterization of epitopes and antigenic differentiation of Australian strains.

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 S1 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.

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.

Immune responses to structural proteins of avian infectious bronchitis virus

Chickens were vaccinated with live and inactivated infectious bronchitis virus (IBV), and antibody responses to the individual structural proteins, S1, S2, M and N, followed by ELISA and western blotting. All four structural proteins elicited an antibody response in chicks vaccinated with either live or inactivated IBV. The S1, S2 and N proteins elicited similar titres of antibodies following vaccination with live IBV, whereas the M glycoprotein elicited significantly lower titres. Time of appearance and the course of development of the S1, S2 and N ELISA antibodies were similar, being first detected 2 weeks after vaccination and coincided with appearance of virus neutralizing antibodies. The M antibodies were first detected 4 weeks after vaccination. S1, S2, and N antibody titres were significantly higher in chicks vaccinated at 14 days of age than in chicks vaccinated at either 1 or 7 days of age, and reached maximum levels 4 weeks after the second vaccination. The S1, S2 and N proteins induced cross-reactive antibodies, whereas the M glycoprotein induced antibodies of limited cross-reactivity. Titres of cross-reactive N antibodies were higher than titres of cross-reactive S1 and S2 antibodies, which were similar. Epitopes on the N and S2 proteins that gave rise to cross-reactive antibodies showed the same degree of conservation, whereas the cross-reactive S1 epitopes were marginally less conserved. Vaccination with inactivated virus induced significantly lower antibody titres and at least three vaccinations were necessary for induction of S1, S2, N and M antibodies in all chicks. The S2 glycoprotein was the most immunogenic structural protein following vaccination with inactivated virus. All four proteins induced cell-mediated immune responses in chicks vaccinated with live IBV as determined by a delayed type hypersensitivity response.