Newcastle Disease Virus (NDV), genus Avulavirus of the family Paramyxoviridae, is also known as avian paramyxovirus type 1. It causes the notifiable Newcastle Disease or atypical fowl plague in poultry. NDV are classified depending on their intracerebal pathogenicity index (ICPI) into different pathotypes (lentogenic with an ICPI of 0-0.7, mesogenic with an ICPI of 0.7-1.5 or velogenic with an ICPI of 1.5-2.0). Virus isolates from poultry with an ICPI in day-old chickens greater than 0.7 have to be notified and are followed by disease control activities. It has been demonstrated that the amino acid sequence at the proteolytic cleavage site of the fusion protein is an important determinant of virulence, but may not be sufficient for velogenic NDV. Since the advent of reverse genetics for NDV, it is now possible and an important focus of current investigations elucidate at the molecular level causes for the different pathogenicity of NDV strains.
To combat Newcastle Disease, vaccination is compulsory, e.g. in Germany. Live or inactivated vaccines of a lentogenic NDV are used by mass application (via spray or drinking water). Qualification of the NDV genome to express additionally inserted foreign genes has been shown and it makes NDV interesting for the development of bivalent vaccines, especially for a vaccine against Newcastle Disease and highly pathogenic avian influenza (HPAI). Such a virus, which expresses only one or two proteins of HPAIV could be used as mass applicable marker vaccine.
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.
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.
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