The purpose of this study is to characterise Australian isolates of bovine enterovirus (BEV) and develop a suitable isolate as a replication-limited vaccine vector. Advantages of using BEV as a vector are that it both elicits mucosal immunity and has naturally occurring temperature stable isolates so that a BEV vector could be administered orally to elicit a protective immune response in the host and should not require cold storage to maintain vaccine efficacy. Furthermore, wildtype BEV causes no or only mild clinical symptoms in its host and if BEV is used as a vaccine vector, reversion to wildtype phenotype would not cause deleterious effects in vaccinated cattle. To date many of the viruses used as vaccine vectors are produced by modifying the structural proteins of the virion so that they contain heterologous sequences. However, each of the four BEV structural proteins are essential and it is not possible to insert large sequences without disrupting the virion. While this study looks at potential insertion sites within the BEV virion, the main focus for the development of BEV as a vaccine vector is through using a replication-limited BEV vector. The development of a replication-limited vector requires the deletion of an essential viral gene that is then replaced in vitro using an expression vector. When the replication-limited vector and its complementing expression cassette are co-transfected into a permissive cell line all the proteins required for viral assembly are produced but only replication deficient genomes are available to be encapsidated. The physically intact but replication deficient viral particles produced in vitro can then infect permissive cells in vivo, resulting in the production of all but the deleted viral protein. Moreover, the deleted portion of the viral genome can be replaced with heterologous sequences within the replication-limited BEV vector. These heterologous sequences can then be expressed in vivo where they can be recognised by the host immune system. Three BEV isolates representing the Australian subserotypes were used in this study: K2577, SL305 and 66/27. The full-length sequences of K2577 and SL305 were obtained as well as partial sequence from the third isolate, 66/27. Sequence homology and phylogenetic analysis showed all three isolates were more closely related to BEV-1 subserotypes than BEV-2. This is the first report to indicate that Australian BEV isolates can be classified as BEV-1. Analysis of the 5’-untranslated region (5’-UTR) indicated that BEV isolates were recombinants with each other and that these recombinant regions correspond to the duplicated cloverleaf structure which is present in BEV 5’-UTR but absent from other enteroviruses. While BEV was initially reported to be stable at higher temperatures, later studies showed that this property varied between isolates and this is also true of the three isolates used in this study. Since it is important not only to ensure that the isolate used as a vaccine vector is temperature stable but also the resulting vaccine vector, the molecular basis of BEV temperature stability was also studied. Using sequence data from the Australian isolates, regions of variation were located and hybrid BEV created. Unfortunately, all of the hybrid BEV produced in this study were non-infectious and could not be used to for further characterisation of BEV temperature stability. Preparatory to constructing replication-limited BEV, a system for full-length amplification of BEV was developed. By including sequences for the bacterial promoter T7 on the positive sense primer used for full-length amplification of BEV, it was possible to prepare full-length transcripts of the amplified product and these were shown to produce infectious BEV particles when transfected into to cell lines that supported BEV growth. Subsequent cloning of the K2577 amplification product resulted in infectious clones for this BEV isolate and these clones were used to prepare replication-limited BEV constructs. To test the replication-limited system BEV structural genes were replaced with a reporter gene to produce replication deficient infectious clones. Complementary constructs containing only the deleted structural genes were also prepared to express the deleted genes. While it was expected that these expression vector would be able to complement the replication deficient BEV in vivo, co-transfection of the replication-limited construct with its complementing expression vector did not produce viable BEV.

The purpose of this study is to characterise Australian isolates of bovine enterovirus (BEV) and develop a suitable isolate as a replication-limited vaccine vector. Advantages of using BEV as a vector are that it both elicits mucosal immunity and has naturally occurring temperature stable isolates so that a BEV vector could be administered orally to elicit a protective immune response in the host and should not require cold storage to maintain vaccine efficacy. Furthermore, wildtype BEV causes no or only mild clinical symptoms in its host and if BEV is used as a vaccine vector, reversion to wildtype phenotype would not cause deleterious effects in vaccinated cattle. To date many of the viruses used as vaccine vectors are produced by modifying the structural proteins of the virion so that they contain heterologous sequences. However, each of the four BEV structural proteins are essential and it is not possible to insert large sequences without disrupting the virion. While this study looks at potential insertion sites within the BEV virion, the main focus for the development of BEV as a vaccine vector is through using a replication-limited BEV vector. The development of a replication-limited vector requires the deletion of an essential viral gene that is then replaced in vitro using an expression vector. When the replication-limited vector and its complementing expression cassette are co-transfected into a permissive cell line all the proteins required for viral assembly are produced but only replication deficient genomes are available to be encapsidated. The physically intact but replication deficient viral particles produced in vitro can then infect permissive cells in vivo, resulting in the production of all but the deleted viral protein. Moreover, the deleted portion of the viral genome can be replaced with heterologous sequences within the replication-limited BEV vector. These heterologous sequences can then be expressed in vivo where they can be recognised by the host immune system. Three BEV isolates representing the Australian subserotypes were used in this study: K2577, SL305 and 66/27. The full-length sequences of K2577 and SL305 were obtained as well as partial sequence from the third isolate, 66/27. Sequence homology and phylogenetic analysis showed all three isolates were more closely related to BEV-1 subserotypes than BEV-2. This is the first report to indicate that Australian BEV isolates can be classified as BEV-1. Analysis of the 5’-untranslated region (5’-UTR) indicated that BEV isolates were recombinants with each other and that these recombinant regions correspond to the duplicated cloverleaf structure which is present in BEV 5’-UTR but absent from other enteroviruses. While BEV was initially reported to be stable at higher temperatures, later studies showed that this property varied between isolates and this is also true of the three isolates used in this study. Since it is important not only to ensure that the isolate used as a vaccine vector is temperature stable but also the resulting vaccine vector, the molecular basis of BEV temperature stability was also studied. Using sequence data from the Australian isolates, regions of variation were located and hybrid BEV created. Unfortunately, all of the hybrid BEV produced in this study were non-infectious and could not be used to for further characterisation of BEV temperature stability. Preparatory to constructing replication-limited BEV, a system for full-length amplification of BEV was developed. By including sequences for the bacterial promoter T7 on the positive sense primer used for full-length amplification of BEV, it was possible to prepare full-length transcripts of the amplified product and these were shown to produce infectious BEV particles when transfected into to cell lines that supported BEV growth. Subsequent cloning of the K2577 amplification product resulted in infectious clones for this BEV isolate and these clones were used to prepare replication-limited BEV constructs. To test the replication-limited system BEV structural genes were replaced with a reporter gene to produce replication deficient infectious clones. Complementary constructs containing only the deleted structural genes were also prepared to express the deleted genes. While it was expected that these expression vector would be able to complement the replication deficient BEV in vivo, co-transfection of the replication-limited construct with its complementing expression vector did not produce viable BEV.

The purpose of this study is to characterise Australian isolates of bovine enterovirus (BEV) and develop a suitable isolate as a replication-limited vaccine vector. Advantages of using BEV as a vector are that it both elicits mucosal immunity and has naturally occurring temperature stable isolates so that a BEV vector could be administered orally to elicit a protective immune response in the host and should not require cold storage to maintain vaccine efficacy. Furthermore, wildtype BEV causes no or only mild clinical symptoms in its host and if BEV is used as a vaccine vector, reversion to wildtype phenotype would not cause deleterious effects in vaccinated cattle. To date many of the viruses used as vaccine vectors are produced by modifying the structural proteins of the virion so that they contain heterologous sequences. However, each of the four BEV structural proteins are essential and it is not possible to insert large sequences without disrupting the virion. While this study looks at potential insertion sites within the BEV virion, the main focus for the development of BEV as a vaccine vector is through using a replication-limited BEV vector. The development of a replication-limited vector requires the deletion of an essential viral gene that is then replaced in vitro using an expression vector. When the replication-limited vector and its complementing expression cassette are co-transfected into a permissive cell line all the proteins required for viral assembly are produced but only replication deficient genomes are available to be encapsidated. The physically intact but replication deficient viral particles produced in vitro can then infect permissive cells in vivo, resulting in the production of all but the deleted viral protein. Moreover, the deleted portion of the viral genome can be replaced with heterologous sequences within the replication-limited BEV vector. These heterologous sequences can then be expressed in vivo where they can be recognised by the host immune system. Three BEV isolates representing the Australian subserotypes were used in this study: K2577, SL305 and 66/27. The full-length sequences of K2577 and SL305 were obtained as well as partial sequence from the third isolate, 66/27. Sequence homology and phylogenetic analysis showed all three isolates were more closely related to BEV-1 subserotypes than BEV-2. This is the first report to indicate that Australian BEV isolates can be classified as BEV-1. Analysis of the 5’-untranslated region (5’-UTR) indicated that BEV isolates were recombinants with each other and that these recombinant regions correspond to the duplicated cloverleaf structure which is present in BEV 5’-UTR but absent from other enteroviruses. While BEV was initially reported to be stable at higher temperatures, later studies showed that this property varied between isolates and this is also true of the three isolates used in this study. Since it is important not only to ensure that the isolate used as a vaccine vector is temperature stable but also the resulting vaccine vector, the molecular basis of BEV temperature stability was also studied. Using sequence data from the Australian isolates, regions of variation were located and hybrid BEV created. Unfortunately, all of the hybrid BEV produced in this study were non-infectious and could not be used to for further characterisation of BEV temperature stability. Preparatory to constructing replication-limited BEV, a system for full-length amplification of BEV was developed. By including sequences for the bacterial promoter T7 on the positive sense primer used for full-length amplification of BEV, it was possible to prepare full-length transcripts of the amplified product and these were shown to produce infectious BEV particles when transfected into to cell lines that supported BEV growth. Subsequent cloning of the K2577 amplification product resulted in infectious clones for this BEV isolate and these clones were used to prepare replication-limited BEV constructs. To test the replication-limited system BEV structural genes were replaced with a reporter gene to produce replication deficient infectious clones. Complementary constructs containing only the deleted structural genes were also prepared to express the deleted genes. While it was expected that these expression vector would be able to complement the replication deficient BEV in vivo, co-transfection of the replication-limited construct with its complementing expression vector did not produce viable BEV.

Im Zuge der Überarbeitung der DVG-Richtlinie zur Prüfung der Viruzidie chemischer Desinfektionsmittel in der Nutztierhaltung wurden BVDV, EAV und PPV auf ihre Eignung als potentielle Prüfviren getestet. Das bisher vorgeschriebene Newcastle-Disease-Virus und das Vacciniavirus sollen mit anderen behüllten Viren wie BVDV oder EAV verglichen werden. Beweggründe für einen möglichen Austausch sind die derzeitige Situation in der Tierseuchenbekämpfung, die Erhöhung der Anwendersicherheit durch Wegfall des zoonotischen Potentials, die einfachere Kultivierung und Handhabung der Prüfviren sowie speziell bei NDV die höhere Aussagekraft der gewonnenen Ergebnisse. Die Desinfektionsmittelversuche wurden gemäß DVG-Richtlinie auf Pappelholzkeimträgern durchgeführt, wobei das jeweilige, mit fetalem Kälberserum vermischte, Virus auf die Keimträger aufgetragen und angetrocknet wurde. Die DVG schreibt eine Trocknung im Brutschrank von 60 Minuten bei 37°C vor. Um die Trocknungsverluste der eingesetzten Viren zu untersuchen, wurden vergleichende Trocknungsversuche wie vorgeschrieben im Brutschrank und im Exsikkator bei Raumtemperatur durchgeführt. Die nach der Trocknung im Brutschrank durchgeführten Desinfektionsmittelversuche wurden mit chemischen Grundsubstanzen kommerziell erhältlicher Desinfektionsmittel durchgeführt. Dabei kamen verschiedene Anwendungskonzentrationen von Ameisensäure, Glutaraldehyd, Natriumhypochlorit, Natronlauge und Peressigsäure zum Einsatz. Bei der vorgeschriebenen Trocknung im Brutschrank kam es zu Titerverlusten von 0,8 bis zu 2,75 log10KID50/ml. Durch eine Trocknung der Holzkeimträger von 30 Minuten bei Raumtemperatur im Exsikkator konnten die Titerverluste auf 0,3 bis 1,0 log10KID50/ml reduziert werden. In den nachfolgenden Desinfektionsversuchen zeigte sich die besonders hohe Tenazität von PPV. Es war den eingesetzten Desinfektionsmitteln gegenüber deutlich resistenter als alle anderen untersuchten Viren. In den Trocknungsversuchen zeigte PPV mit Abstand die niedrigsten Titerverluste. Mit BVDV und EAV konnten zwar ausreichend hohe Titer erzielt werden, allerdings waren die Trocknungsverluste beider Viren sehr hoch. In den Keimträgerversuchen konnte nur in wenigen Versuchen eine Titerreduktion von mehr als 3 Logarithmusstufen erreicht werden. Hier könnte zukünftig die Trocknung im Exsikkator Abhilfe schaffen, um die Trocknungsverluste zu minimieren und eine höhere Titerreduktion zu ermöglichen. Die Ergebnisse einer früheren Arbeit zeigen identische Ergebnisse von NDV und BVDV im Keimträgertest. Ein Ersatz von NDV durch BVDV ist somit zu empfehlen. Eine Verwendung der untersuchten Viren gemäß den derzeitigen DVG-Richtlinien ist möglich, allerdings müssten im Zuge der weiteren Harmonisierung von CEN- und DVG-Richtlinie die Kontrolltiter entsprechend erhöht werden, um die von der CEN geforderte Titerreduktion von vier Logarithmusstufen für eine vollständige Virusinaktivierung einzuhalten. Die Vermehrung der untersuchten Viren zu höheren Ausgangs-, bzw. Kontrolltitern sollte daher Gegenstand weiterer Forschungsarbeit sein. Einer weiteren Verwendung der bisherigen Prüfviren BEV und REOV steht nichts im Wege. Aufgrund der Ergebnisse der vergleichenden Trocknungsversuche wird für alle untersuchten Viren zukünftig eine 30 minütige Trocknung im Exsikkator empfohlen.

Im Zuge der Überarbeitung der DVG-Richtlinie zur Prüfung der Viruzidie chemischer Desinfektionsmittel in der Nutztierhaltung wurden BVDV, EAV und PPV auf ihre Eignung als potentielle Prüfviren getestet. Das bisher vorgeschriebene Newcastle-Disease-Virus und das Vacciniavirus sollen mit anderen behüllten Viren wie BVDV oder EAV verglichen werden. Beweggründe für einen möglichen Austausch sind die derzeitige Situation in der Tierseuchenbekämpfung, die Erhöhung der Anwendersicherheit durch Wegfall des zoonotischen Potentials, die einfachere Kultivierung und Handhabung der Prüfviren sowie speziell bei NDV die höhere Aussagekraft der gewonnenen Ergebnisse. Die Desinfektionsmittelversuche wurden gemäß DVG-Richtlinie auf Pappelholzkeimträgern durchgeführt, wobei das jeweilige, mit fetalem Kälberserum vermischte, Virus auf die Keimträger aufgetragen und angetrocknet wurde. Die DVG schreibt eine Trocknung im Brutschrank von 60 Minuten bei 37°C vor. Um die Trocknungsverluste der eingesetzten Viren zu untersuchen, wurden vergleichende Trocknungsversuche wie vorgeschrieben im Brutschrank und im Exsikkator bei Raumtemperatur durchgeführt. Die nach der Trocknung im Brutschrank durchgeführten Desinfektionsmittelversuche wurden mit chemischen Grundsubstanzen kommerziell erhältlicher Desinfektionsmittel durchgeführt. Dabei kamen verschiedene Anwendungskonzentrationen von Ameisensäure, Glutaraldehyd, Natriumhypochlorit, Natronlauge und Peressigsäure zum Einsatz. Bei der vorgeschriebenen Trocknung im Brutschrank kam es zu Titerverlusten von 0,8 bis zu 2,75 log10KID50/ml. Durch eine Trocknung der Holzkeimträger von 30 Minuten bei Raumtemperatur im Exsikkator konnten die Titerverluste auf 0,3 bis 1,0 log10KID50/ml reduziert werden. In den nachfolgenden Desinfektionsversuchen zeigte sich die besonders hohe Tenazität von PPV. Es war den eingesetzten Desinfektionsmitteln gegenüber deutlich resistenter als alle anderen untersuchten Viren. In den Trocknungsversuchen zeigte PPV mit Abstand die niedrigsten Titerverluste. Mit BVDV und EAV konnten zwar ausreichend hohe Titer erzielt werden, allerdings waren die Trocknungsverluste beider Viren sehr hoch. In den Keimträgerversuchen konnte nur in wenigen Versuchen eine Titerreduktion von mehr als 3 Logarithmusstufen erreicht werden. Hier könnte zukünftig die Trocknung im Exsikkator Abhilfe schaffen, um die Trocknungsverluste zu minimieren und eine höhere Titerreduktion zu ermöglichen. Die Ergebnisse einer früheren Arbeit zeigen identische Ergebnisse von NDV und BVDV im Keimträgertest. Ein Ersatz von NDV durch BVDV ist somit zu empfehlen. Eine Verwendung der untersuchten Viren gemäß den derzeitigen DVG-Richtlinien ist möglich, allerdings müssten im Zuge der weiteren Harmonisierung von CEN- und DVG-Richtlinie die Kontrolltiter entsprechend erhöht werden, um die von der CEN geforderte Titerreduktion von vier Logarithmusstufen für eine vollständige Virusinaktivierung einzuhalten. Die Vermehrung der untersuchten Viren zu höheren Ausgangs-, bzw. Kontrolltitern sollte daher Gegenstand weiterer Forschungsarbeit sein. Einer weiteren Verwendung der bisherigen Prüfviren BEV und REOV steht nichts im Wege. Aufgrund der Ergebnisse der vergleichenden Trocknungsversuche wird für alle untersuchten Viren zukünftig eine 30 minütige Trocknung im Exsikkator empfohlen.