The molecular basis of the pathogenicity of Newcastle disease virus in chickens

Abstract

Newcastle disease (ND) is a highly pathogenic disease of poultry and is caused by virulent strains of Newcastle disease virus (NDV). From 1998-2002 there were outbreaks of ND in Australia which resulted in significant disruptions to the poultry industry. In some of these outbreaks however, the clinical signs observed in the infected birds did not appear to correlate with the World Organisation for Animal Health’s definition of a virulent virus, which is based on the molecular sequence at the fusion protein cleavage site. In one particular outbreak at Meredith, Victoria, in 2002, a virulent virus was isolated, despite only a minimal increase in mortalities on the property. Therefore, this thesis has attempted to determine whether, in addition to the fusion protein cleavage site, there are other molecular determinants of pathogenicity for NDV. The pathogenicity of the Meredith/02 virus was first characterised by experimental infection of specific pathogen free (SPF) chickens. The Meredith/02 virus was compared with an avirulent virus (Peats Ridge/98) and two other virulent viruses (Herts 33/56 and Texas GB) using clinical evaluation, histopathology, immunohistochemistry and molecular techniques. The Meredith/02 virus showed minimal clinical signs in a small number of birds and no mortalities. The birds infected with Herts 33/56 and Texas GB were all euthanased at day 2 post inoculation and day 5 post inoculation respectively. The minimal pathogenicity of the Meredith/02 virus was associated with decreased virus replication and antigen distribution in a number of tissues when compared with the Herts 33/56 and Texas GB viruses. Further characterisation of the Meredith/02 virus showed that it contained a virulent fusion protein cleavage site motif of 112RRQRRF117, which is exactly the same as the cleavage site of Herts 33/56. The mean death time in eggs classified the virus as a mesogenic NDV. Sequence analysis showed a number of amino acid differences throughout the genomes of the four viruses studied, however none of these differences were in key areas such as glycosylation sites. The Meredith/02 virus was also shown to replicate well in embryonated eggs, throughout the chorioallantoic membrane and internal organs of the embryo, including the lung, liver and kidneys. This is consistent with other virulent NDVs. The V protein of the Meredith/02 virus was investigated for its role in potential attenuation of the virus via modulation of the host innate immune response. However there was no difference found in the ability of the Meredith/02 V protein to antagonise type I interferon in-vitro when compared with the Herts 33/56 virus. In an attempt to analyse the viral replication complex, to identify a specific protein that may be involved with the minimal pathogenicity of the Meredith/02 virus, the transcription gradient of the virus was characterized. It was found that the Meredith/02 virus has an increased transcription gradient when compared with the Herts 33/56 virus. The gradient of the Meredith/02 virus was particularly steep at the N-P junction, with particularly low levels of the P gene transcribed at 24 hours. However, gene start and end sequences at this location did not vary between the two viruses, thereby indicating that the N and P proteins are less likely to be associated with the steepened gradient. Instead, this suggests a possible role for the large polymerase protein in decreasing transcription. Whilst this research has not yet identified specific molecular sequences responsible for the minimal pathogenicity of the Meredith/02 virus despite its virulent fusion protein cleavage site, it has focused the investigation on components of the viral replication complex. Therefore directions for further research include investigating the role of the replication complex, in particular, the large polymerase (L) gene in the pathogenicity of Australian NDVs. This could also incorporate further work on the individual proteins via the use of minigenome assays, or by utilising reverse genetics and full-length virus clones. Additional transcriptional profiles of other mesogenic viruses could also be analysed. It would also be interesting to compare the pathogenicity of the Meredith/02 virus with other viruses from the 1998-2002 Australian outbreaks in an experimental setting. The outcomes from this work have provided greater insight into an Australian NDV which until now has not been well characterised. This research is also relevant to the broader group of mesogenic NDVs which are not easily classified according to their fusion protein cleavage sites.