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Conceptualization, R.D.; methodology, R.D. and J.K.; validation, N.O., N.N., S.H. and D.-Y.O.; formal analysis, D.-Y.O.; investigation, R.D. and J.K.; resources, H.L., N.O. and N.N.; data curation, R.D.; writing—original draft preparation, R.D.; writing—review and editing, R.D., S.H., D.-Y.O., N.N., N.O. and J.K.; visualization, R.D., J.K. and D.-Y.O.; supervision, R.D., N.N., N.O. and H.L.; project administration, R.D. All authors have read and agreed to the published version of the manuscript.
Figure 1. BoDV-1 vaccines used in Germany. (a) BD live virus vaccine “Dessau”, instructions for use (in German), package, and five vials containing lyophilized rabbit brain with BoDV-1 “Dessau” (cluster 3). Shown is the penultimate batch produced in November 1990, which was sequenced in this study (b) Label (in German) of the Zwick vaccine “Giessen” containing strain V from 1948, which was sequenced in this study (cluster 4).
Figure 1. BoDV-1 vaccines used in Germany. (a) BD live virus vaccine “Dessau”, instructions for use (in German), package, and five vials containing lyophilized rabbit brain with BoDV-1 “Dessau” (cluster 3). Shown is the penultimate batch produced in November 1990, which was sequenced in this study (b) Label (in German) of the Zwick vaccine “Giessen” containing strain V from 1948, which was sequenced in this study (cluster 4).
Figure 2. Phylogenetic analyses of the vaccine viruses Zwick “Giessen” and “Dessau” (red squares). The tree comprises 41 full genomes (8823 bp) of BoDV-1 (MEGA X, Neighbor-Joining method, p-distance model, pairwise deletion option, 1000 replicates, bootstrap values ≥ 80).
Figure 2. Phylogenetic analyses of the vaccine viruses Zwick “Giessen” and “Dessau” (red squares). The tree comprises 41 full genomes (8823 bp) of BoDV-1 (MEGA X, Neighbor-Joining method, p-distance model, pairwise deletion option, 1000 replicates, bootstrap values ≥ 80).
Figure 3. Inactivation kinetics of BoDV-1 “Dessau” during binary ethylenimine treatment (left) and storage at room temperature (right).
Figure 3. Inactivation kinetics of BoDV-1 “Dessau” during binary ethylenimine treatment (left) and storage at room temperature (right).
Figure 4. Titration of infectious dose of OL-cell-culture-derived BoDV-1 (virus V/B) in rabbits. Shown are the kinetics of body weights, immunofluorescent (IF) antibodies and neutralizing (NT) antibodies following infection challenge with 105 FFU OL-derived BoDV-1, 104 FFU OL-derived BoDV-1, 103 FFU OL-derived BoDV-1, 102 FFU OL-derived BoDV-1, and 101 FFU OL-derived BoDV-1. Dashed lines, IF and NT assay limits of detection; †, animal euthanized before succumbing to lethal illness.
Figure 4. Titration of infectious dose of OL-cell-culture-derived BoDV-1 (virus V/B) in rabbits. Shown are the kinetics of body weights, immunofluorescent (IF) antibodies and neutralizing (NT) antibodies following infection challenge with 105 FFU OL-derived BoDV-1, 104 FFU OL-derived BoDV-1, 103 FFU OL-derived BoDV-1, 102 FFU OL-derived BoDV-1, and 101 FFU OL-derived BoDV-1. Dashed lines, IF and NT assay limits of detection; †, animal euthanized before succumbing to lethal illness.
Figure 5. Titration of infectious dose of brain-derived BoDV-1 (strain V/B) in rabbits. Shown are the kinetics of body weight changes, immunofluorescent (IF) antibody titers and neutralizing (NT) antibody titers following infection challenge with 105 FFU brain-derived BoDV-1, 104 FFU brain-derived BoDV-1, 103 brain-derived BoDV-1, 102 FFU brain-derived BoDV-1, and 101 FFU brain-derived BoDV-1. Dashed lines, IF and NT assay limits of detection; †, animal euthanized before succumbing to lethal illness.
Figure 5. Titration of infectious dose of brain-derived BoDV-1 (strain V/B) in rabbits. Shown are the kinetics of body weight changes, immunofluorescent (IF) antibody titers and neutralizing (NT) antibody titers following infection challenge with 105 FFU brain-derived BoDV-1, 104 FFU brain-derived BoDV-1, 103 brain-derived BoDV-1, 102 FFU brain-derived BoDV-1, and 101 FFU brain-derived BoDV-1. Dashed lines, IF and NT assay limits of detection; †, animal euthanized before succumbing to lethal illness.
Figure 6. Titration of vaccine doses: Neutralizing antibody titers in vaccinated rabbits. BoDV-1 live vaccine “Dessau” containing 103 FFU and 101 FFU per dose (a) were compared with batches of ethylenimine-inactivated BoDV-1 “Dessau” vaccines containing either (b) no adjuvant and 107, 105, 103, 101 FFU BoDV-1 or (c) ISA25 (mineral oil-based o/w) adjuvant and 107, 105, 103, 101 FFU BoDV-1 before inactivation. Shown is the mean ± SEM of neutralizing antibody titers (ND50) in rabbits after the first, second (3 weeks after 1st) and third (16 weeks after 2nd) vaccine administration; titer determinations were based on BoDV-1 strain V/B. Dashed lines, NT assay limit of detection.
Figure 6. Titration of vaccine doses: Neutralizing antibody titers in vaccinated rabbits. BoDV-1 live vaccine “Dessau” containing 103 FFU and 101 FFU per dose (a) were compared with batches of ethylenimine-inactivated BoDV-1 “Dessau” vaccines containing either (b) no adjuvant and 107, 105, 103, 101 FFU BoDV-1 or (c) ISA25 (mineral oil-based o/w) adjuvant and 107, 105, 103, 101 FFU BoDV-1 before inactivation. Shown is the mean ± SEM of neutralizing antibody titers (ND50) in rabbits after the first, second (3 weeks after 1st) and third (16 weeks after 2nd) vaccine administration; titer determinations were based on BoDV-1 strain V/B. Dashed lines, NT assay limit of detection.
Figure 7. Neutralizing (NT) antibody titer kinetics in rabbits vaccinated with BoDV-1 live vaccine “Dessau” after (a) heterologous challenge infection with BoDV-1 V/B or (b) homologous challenge infection with the BoDV-1 vaccine virus “Dessau”. Body weight development after the respective challenge infections is shown in (c,d). Dashed lines, IF and NT assay limits of detection; †, animal euthanized before succumbing to lethal illness.
Figure 7. Neutralizing (NT) antibody titer kinetics in rabbits vaccinated with BoDV-1 live vaccine “Dessau” after (a) heterologous challenge infection with BoDV-1 V/B or (b) homologous challenge infection with the BoDV-1 vaccine virus “Dessau”. Body weight development after the respective challenge infections is shown in (c,d). Dashed lines, IF and NT assay limits of detection; †, animal euthanized before succumbing to lethal illness.
Figure 8. NT antibody kinetics in rabbits after vaccination with inactivated vaccines based on BoDV-1 strain V/B, following three doses of nonadjuvanted vaccine (a) or of ISA25-adjuvanted vaccine (b). Body weights after challenge with BoDV-1 V/B in animals vaccinated with nonadjuvanted inactivate (c) and in the animals vaccinated with ISA25-adjuvanted inactivated vaccine (d). NT antibody titers [ND50] in the nonadjuvanted group after challenge infection (e) and in the ISA25-adjuvanted group after challenge infection (f). Dashed lines, IF and NT assay limits of detection; †, animal euthanized before succumbing to lethal illness.
Figure 8. NT antibody kinetics in rabbits after vaccination with inactivated vaccines based on BoDV-1 strain V/B, following three doses of nonadjuvanted vaccine (a) or of ISA25-adjuvanted vaccine (b). Body weights after challenge with BoDV-1 V/B in animals vaccinated with nonadjuvanted inactivate (c) and in the animals vaccinated with ISA25-adjuvanted inactivated vaccine (d). NT antibody titers [ND50] in the nonadjuvanted group after challenge infection (e) and in the ISA25-adjuvanted group after challenge infection (f). Dashed lines, IF and NT assay limits of detection; †, animal euthanized before succumbing to lethal illness.
Figure 9. NT antibody kinetics in horses after vaccination with inactivated vaccines based on BoDV-1 “Dessau”, following three applications of the nonadjuvanted vaccine (a), or ISA25-adjuvanted vaccine (b). Rectal temperatures after the first vaccine administration in horses vaccinated with the nonadjuvanted inactivated vaccine (c) and in the group vaccinated with the ISA25-adjuvanted inactivated vaccine (d). One horse developed colic and had to be taken out of the trial. Dashed lines, NT assay limit of detection.
Figure 9. NT antibody kinetics in horses after vaccination with inactivated vaccines based on BoDV-1 “Dessau”, following three applications of the nonadjuvanted vaccine (a), or ISA25-adjuvanted vaccine (b). Rectal temperatures after the first vaccine administration in horses vaccinated with the nonadjuvanted inactivated vaccine (c) and in the group vaccinated with the ISA25-adjuvanted inactivated vaccine (d). One horse developed colic and had to be taken out of the trial. Dashed lines, NT assay limit of detection.
Table 1. Overview of amino acid substitutions present only in strain V viruses, but not in other BoDV-1.
Table 1. Overview of amino acid substitutions present only in strain V viruses, but not in other BoDV-1.
Number of MutationsTable 2. Overview of amino acid substitutions present only in BoDV-1 “Dessau”, but not in other BoDV-1.
Table 2. Overview of amino acid substitutions present only in BoDV-1 “Dessau”, but not in other BoDV-1.
Number of MutationsTable 3. Neutralizing antibody titers after vaccination with BoDV-1 live vaccine “Dessau” in horses already vaccinated in the years prior.
Table 3. Neutralizing antibody titers after vaccination with BoDV-1 live vaccine “Dessau” in horses already vaccinated in the years prior.
HorseWeeks Post Vaccination012345810131722I<4 175- 28020--<4-<4-II<417-4035--4-4-III<410--16--<4-<4-IV<48--32--4-<4-V<415 158--8-6-VI<4<4-<4<4--<4-<4-VII<4--2511--<4-<4-VIII<4--3028--4-6-IX<480-40---32-20-X<416-80--- -<4-XI832-32---32-24-XII<4<4-4---4-<4-XIII163235------16-XIV16163220-----<4-XV<4311818-----<4-XVI<4<4---------XVII<430801618----<4-XVIII<432853520----10-XIX4803221-8---8-XX<4152730-8---8-XXI<4153117-<4---<4-XXII<4<4<4-<4-<4-<4-<4XXIII<4<4<4-<4-<4-<4-<4XXIV<4<4<4-<4-<4-<4-<4XXV<4<4<4-<4-<4-<4-<4XXVI<4<4<4-10-<4-<4-<4XXVII<4<4<4-<4-<4-<4-<4XXVIII808080-300-520-640-640XXiX<48080-80-16-4-<4XXX<4<4<4-<4-<4-<4-<4XXXI<416080-80-8-8-8Table 4. Neutralizing antibody titers after first vaccination with BoDV-1 live vaccine “Dessau” in horses.
Table 4. Neutralizing antibody titers after first vaccination with BoDV-1 live vaccine “Dessau” in horses.
HorseWeeks Post Vaccination012345810131722XXXII<4 1<4- 2----<4-<4-XXXIII<4<4-<4-4-<4-<4-XXXIV<4<4860-40-<4-<4-XXXV<4<4<4-<4-16-<4-<4-XXXVI<4<4<4<4-<4-<4-<4-XXXVII<4444-30---<4-XXXVIII<4<4-32 --<4-<4-
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