Molecular determinants of NDV virulence
Reverse Genetics allows site-specific alterations in the genomes of RNA-viruses by mutagenesis of single nucleotides or insertion of foreign genes. These approaches are being used to study the molecular basis of NDV virulence. It is known that the amino acid sequence at the proteolytic cleavage site of the F protein has a major impact on NDV pathogenicity. Single basic amino acids (GRQGR*L) at the carboxyterminus of the F2 subunit and a leucine residue at the aminoterminus of the F1 subunit are typical for lentogenic strains. Mesogenic und velogenic virus strains specify two pairs of basic amino acids and a phenylalanine (RR/KQR/KR*F) in this region. This could be confirmed by isolation of respective recombinant viruses. However, it was also clear that this is not the sole determining feature of pathogenicity. A recombinant virus specifying RRQKR*F in a lentogenic strain background exhibited a significant increase in pathogenicity as demonstrated by syncytia-formation in cell culture and an ICPI of 1.3. However, no transformation to a velogenic phenotype was observed. By exchange of the two surface glycoproteins F and HN of lentogenic clone 30 versus those of a mesogenic NDV a recombinant virus was generated which, despite the presence of a polybasic F-protein cleavage site, exhibits a lentogenic phenotype. This reinforces the finding that additional pathogenicity determinants exist.
Furthermore we showed that in naturally occurring NDV the variability in the length of the HN open reading frames had no influence on pathogenicity.
Development of a mass applicable marker vaccine against NDV and avian influenza
Avian influenza is a zoonotic disease with serious economic consequences. Although vaccines for poultry are available they have considerable disadvantages with regard to application, onset of immunity, and efficacy of production which limit their use.
The aim of this project is the development of an NDV vector vaccine for protection of poultry from Newcastle Disease as well as highly pathogenic avian influenza. Such a vaccine would be mass applicable by spray or drinking water. The insertion of an influenza-derived open reading frame into the NDV genome should be done in a way which allowed rapid substitution to adapt vaccine virus to the changing field situation. To maximize the expression level of of the foreign protein, different insertion sites within the NDV genome will be used. Vaccine candidates are investigated in vitro and in vivo for their pathogenicity, stability, immunological response and prevention of virus shedding after immunization as well as after challenge infection. Qualification of vaccine virus as a marker vaccine will be confirmed by appropriate diagnostic tests.