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Cited by 12Animal Virus Infections StudiesVirology and Viral Diseasesvaccines and immunoinformatics approaches

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Evaluating the Breadth of Neutralizing Antibody Responses Elicited by Infectious Bursal Disease Virus Genogroup A1 Strains Using a Novel Chicken B-Cell Rescue System and Neutralization Assay

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Reddy, Vishwanatha R. A. P., Nazki, Salik, Brodrick, Andrew J., Asfor, Amin, Urbaniec, Joanna, Morris, Yasmin, Broadbent, Andrew J.·2022·Journal of Virology
DOI 10.1128/jvi.01255-22PMC PMC9517715

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Abstract

There is a need to evaluate the ability of vaccines to neutralize diverse IBDV genogroups and to better understand the relationship between HVR sequence, structure, and antigenicity. Here, we used a chicken B-cell line to rescue a panel of chimeric IBDVs with the HVR from seven diverse IBDV field strains and to conduct neutralization assays and protein modeling.

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Figures

Figure 1 DT40 cells can be used to quantify the titer of IBDV, quantify the titer of anti-IBDV serum neutralising antibodies, and rescue a molecular clone of IBDV. The vv IBDV field strain UK66l was serially diluted ten-fold from l:100to 1:100,000, and the diluted stocks were frozen at -80 °C. Each diluted stock was subsequently thawed and subject to titration by TCID50 in DT40 cells, and by EID50 in embyronated chicken eggs, and a linear regression analysis was performed (A). Hyperimmune sera from birds inoculated with the IBDV vaccine strain 2512 (genogroup A1) was obtained from Charles River. The serum was heat inactivated, serially diluted two-fold, and mixed with 100 TCID50 of UK66l. The mixture was added to DT40 cells in quadruplicate, and after 3 days, the wells were fixed and stained with an antibody against the IBDV VP3 protein and a secondary antibody conjugated to a fluorophore. The wells were either scored positive or negative for the presence of IBDV antigen by immunofluorescence microscopy, and the percentage of positive cells in each well was quantified by flow cytometry to calculate the titer of the neutralising antibodies in the serum. Each point represents the % of VP3 + DT40 cells in one well, the bar represents the mean, and error bars represent the standard deviation of the mean. The horizontal dashed line represents the limit of detection by TCID50 (B). Plasmids pBG98A and pPBG98B were electroporated to DT40 cells, and cell cultures were fed with fresh DT40 cells every 72 hours. At each passage, the supernatant of the cultures was harvested, and serially diluted 10-fold in additional DT40 cells, to determine the titer as described by Reed & Muench. Three biological repeats were titrated and the mean titer plotted for each passage. Error bars represent the standard deviation of the mean (C).
Figure 1 DT40 cells can be used to quantify the titer of IBDV, quantify the titer of anti-IBDV serum neutralising antibodies, and rescue a molecular clone of IBDV. The vv IBDV field strain UK66l was serially diluted ten-fold from l:100to 1:100,000, and the diluted stocks were frozen at -80 °C. Each diluted stock was subsequently thawed and subject to titration by TCID50 in DT40 cells, and by EID50 in embyronated chicken eggs, and a linear regression analysis was performed (A). Hyperimmune sera from birds inoculated with the IBDV vaccine strain 2512 (genogroup A1) was obtained from Charles River. The serum was heat inactivated, serially diluted two-fold, and mixed with 100 TCID50 of UK66l. The mixture was added to DT40 cells in quadruplicate, and after 3 days, the wells were fixed and stained with an antibody against the IBDV VP3 protein and a secondary antibody conjugated to a fluorophore. The wells were either scored positive or negative for the presence of IBDV antigen by immunofluorescence microscopy, and the percentage of positive cells in each well was quantified by flow cytometry to calculate the titer of the neutralising antibodies in the serum. Each point represents the % of VP3 + DT40 cells in one well, the bar represents the mean, and error bars represent the standard deviation of the mean. The horizontal dashed line represents the limit of detection by TCID50 (B). Plasmids pBG98A and pPBG98B were electroporated to DT40 cells, and cell cultures were fed with fresh DT40 cells every 72 hours. At each passage, the supernatant of the cultures was harvested, and serially diluted 10-fold in additional DT40 cells, to determine the titer as described by Reed & Muench. Three biological repeats were titrated and the mean titer plotted for each passage. Error bars represent the standard deviation of the mean (C).CC BY · PubMed Central
Figure 2 Construction of a panel of chimeric IBDVs with the backbone of the lab adapted PBG98 strain and the HVR from diverse field strains. Reverse genetics plasmids encoding segment A of the lab-adapted strain PBG98 (pPBG98A) were designed where the 333 nucleotides that encode the 111 amino acids (residues 220 to 330) of the HVR were swapped for one of seven field strains (pPBG98A-HVR-VP2s of 7 IBDVs). The plasmids were co-electroporated with the reverse genetics plasmid encoding segment B (pPBG98B) into DT40 cells to rescue the viruses (A). The HVR amino acid sequences from the seven field strains F52-70, Del-E, SHG19, UK66l, M04/09, ITA-04 and Vic-01/94 were aligned with PBG98. The accession numbers and genogroup numbers are given in parenthesis. Conserved residues are depicted as black dots and different residues are highlighted in red (B). The replication kinetics of the seven recombinant chimeric IBDVs was determined in triplicate by titration of infected cell supernatants at 12, 24 and 48 hpi in DT40 cells, expressed as log10 TCID50/mL, and the mean plotted (error bars represent standard deviation of the mean) (C).
Figure 2 Construction of a panel of chimeric IBDVs with the backbone of the lab adapted PBG98 strain and the HVR from diverse field strains. Reverse genetics plasmids encoding segment A of the lab-adapted strain PBG98 (pPBG98A) were designed where the 333 nucleotides that encode the 111 amino acids (residues 220 to 330) of the HVR were swapped for one of seven field strains (pPBG98A-HVR-VP2s of 7 IBDVs). The plasmids were co-electroporated with the reverse genetics plasmid encoding segment B (pPBG98B) into DT40 cells to rescue the viruses (A). The HVR amino acid sequences from the seven field strains F52-70, Del-E, SHG19, UK66l, M04/09, ITA-04 and Vic-01/94 were aligned with PBG98. The accession numbers and genogroup numbers are given in parenthesis. Conserved residues are depicted as black dots and different residues are highlighted in red (B). The replication kinetics of the seven recombinant chimeric IBDVs was determined in triplicate by titration of infected cell supernatants at 12, 24 and 48 hpi in DT40 cells, expressed as log10 TCID50/mL, and the mean plotted (error bars represent standard deviation of the mean) (C).CC BY · PubMed Central
Figure 3 Sequencing analysis of the plasmids and viruses. The nucleotide sequences of the HVRs encoded by the seven plasmids, and present in the seven rescued and DT40-passaged viruses, were compared to the sequences in GenBank (Accession numbers provided) for strains F52-70, Del-E, SHG19, UK66l, M04/09, ITA-04 and Vic-01/94. For each strain, the sequence in GenBank is displayed and conserved residues in the plasmid and the rescued virus are depicted as black dots, and different residues were listed. The four hydrophilic loops (P-BC, P-DE, P-FG, and P-HI), important for antigenicity, are boxed. Mutations previously reported to be involved in the adaptation of IBDV to adherent cell culture (positions 253, 279, 284 and 330) are highlighted in blue, and other common variable positions are shaded in orange.
Figure 3 Sequencing analysis of the plasmids and viruses. The nucleotide sequences of the HVRs encoded by the seven plasmids, and present in the seven rescued and DT40-passaged viruses, were compared to the sequences in GenBank (Accession numbers provided) for strains F52-70, Del-E, SHG19, UK66l, M04/09, ITA-04 and Vic-01/94. For each strain, the sequence in GenBank is displayed and conserved residues in the plasmid and the rescued virus are depicted as black dots, and different residues were listed. The four hydrophilic loops (P-BC, P-DE, P-FG, and P-HI), important for antigenicity, are boxed. Mutations previously reported to be involved in the adaptation of IBDV to adherent cell culture (positions 253, 279, 284 and 330) are highlighted in blue, and other common variable positions are shaded in orange.CC BY · PubMed Central
Figure 4 Structural modelling of the HVRs. The predicted structure of the VP2 of IBDV strain PBG98 was modelled using AlphaFold. Images were generated with PyMol, and the side and end-on (axial) views were displayed. The HVR was depicted as solid grey. The predicted structures of PBG98, F52-70, Del-E, SHG19, UK66l, M04/09, ITA-04, and Vic-01/94 were modelled based on the sequence that were in GenBank. For each virus, the side view of the HVR is shown, with the end-on (axial) view shown as an inset. The structures were compared to the PBG98 HVR structure and amino acid differences highlighted in orange (A). The predicted structures of the HVRs of the DT40-passaged viruses were modelled using AlphaFold, and residues that were different from the GenBank sequences were highlighted in purple (B).
Figure 4 Structural modelling of the HVRs. The predicted structure of the VP2 of IBDV strain PBG98 was modelled using AlphaFold. Images were generated with PyMol, and the side and end-on (axial) views were displayed. The HVR was depicted as solid grey. The predicted structures of PBG98, F52-70, Del-E, SHG19, UK66l, M04/09, ITA-04, and Vic-01/94 were modelled based on the sequence that were in GenBank. For each virus, the side view of the HVR is shown, with the end-on (axial) view shown as an inset. The structures were compared to the PBG98 HVR structure and amino acid differences highlighted in orange (A). The predicted structures of the HVRs of the DT40-passaged viruses were modelled using AlphaFold, and residues that were different from the GenBank sequences were highlighted in purple (B).CC BY · PubMed Central

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Metadata

Authors
Reddy, V. R. A. P., Nazki, S., Brodrick, A. J., Asfor, A., Urbaniec, J., Morris, Y., & Broadbent, A. J.
Affiliations
  • The Pirbright Institute (GB)
  • University of Maryland, College Park (US)
Corresponding author
  • Reddy, Vishwanatha R. A. P.
  • Broadbent, Andrew J.
ORCID
Venue
Journal of Virology
Publisher
American Society for Microbiology
Published
2022-09-28
Volume
96
Issue
18
Pages
e0125522-e0125522
ISSN
  • 0022-538X (ISSN-L)
  • 1098-5514
DOI
10.1128/jvi.01255-22
PMC
PMC9517715
OpenAlex
W4294921245
Cited by
12
References
55
Citations by year
2026: 3 · 2025: 1 · 2024: 2 · 2023: 3 · 2022: 3
FWCI
1.40
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Virology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
UKRI | Biotechnology and Biological Sciences Research Council (BB/S014594/1, BBS/E/I/00001845, BBS/E/I/00007034, BBS/E/I/00007039)
Topics
  • Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Animal Virus Infections Studies
  • Health Sciences › Medicine › Epidemiology › Virology and Viral Diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › vaccines and immunoinformatics approaches
Keywords
infectious bursal disease virus, neutralizing antibodies, antigenic relatedness, vaccine design, immunodominant epitopes, conserved epitopes, protein structure modeling, vaccine screening
MeSH terms
Animals, Epitopes, Australia, Chickens, Genotype, Infectious bursal disease virus, Poultry Diseases, Birnaviridae Infections, Antibodies, Neutralizing

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CoChat. (2026). Citation check: Evaluating the Breadth of Neutralizing Antibody Responses Elicited by Infectious Bursal Disease Virus Genogroup A1 Strains Using a Novel Chicken B-Cell Rescue System and Neutralization Assay. https://app.cochat.ai/papers/10.1128/jvi.01255-22
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Reddy, V. R. A. P., Nazki, S., Brodrick, A. J., Asfor, A., Urbaniec, J., Morris, Y., & Broadbent, A. J. (2022). Evaluating the Breadth of Neutralizing Antibody Responses Elicited by Infectious Bursal Disease Virus Genogroup A1 Strains Using a Novel Chicken B-Cell Rescue System and Neutralization Assay. Journal of Virology. https://doi.org/10.1128/jvi.01255-22

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