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Molecular epidemiology
of Newcastle disease virus In Australia
An analysis of the genome
of Australian Newcastle disease viruses to better
understand the epidemiology of virulent disease outbreaks and their control
by Dr. Allan R. Gould
December 2004
RIRDC Publication No 04/139 RIRDC Project No CSA-11J
Background and methods
The project was initiated
in response to the outbreak of virulent Newcastle disease virus that initially
appeared at Dean Park in NSW in 1998. At that time, it was not known whether
the outbreak was caused by the incursion of an exotic overseas NDV into
Australia or by a virus already present in Australia.
Subsequent to the initial outbreak, virulent virus was also detected at Mangrove Mountain in NSW, also in 1998. This outbreak continued until 2000, even in the presence of a program involving quarantine and slaughter of affected poultry. During the period of this project, virulent NDV and the progenitor virus was also detected in Orchard Hills, Glenorie, Marsden Park, Tamworth, Appin and Rossmore. A separate outbreak occurred at Meredith (Victoria) in 2002 and most recently in Horsley Park, NSW (2002).
The research undertaken aimed to fully characterise the viruses responsible for virulent NDV in the poultry industry in Australia and to develop new and improved diagnostic procedures involving amplification of the genome of the virus responsible for the disease. A procedure was finally adopted whereby it was possible to proceed from infected poultry tissue to the detection and determination of the virulent viral sequences within 12-18 hours using the polymerase chain reaction (PCR) and rapid sequencing methodologies. The virulence of the ND virus was assessed using amino acid sequence parameters of the fusion cleavage signal in the fusion protein. Gene sequence was also determined from other regions of the NDV genome to attempt phylogenetic characterisation of the viruses so that the lineage or ancestry of these viruses could be determined. One such location was obtained which enabled the assignment of NDV to different families within those known to circulate in Australia.
It should be noted that in the context used throughout this report, virulent describes an NDV with a fusion cleavage site of RRQRRF (where R=a basic amino acid); while an avirulent virus is defined as having one or other alterations in the dibasic amino acid motifs (generally to a G residue) and/or the lack of the F amino acid i.e. RRQRGL.
Molecular characterisation
of Australian NDV isolates
To obtain a better understanding
of the viruses responsible for the outbreaks of virulent NDV in Australia
and to set a bench mark for further research studies, the complete genome
of the virulent and avirulent ‘progenitor’ viruses involved in the Dean
Park, Peats Ridge (Mangrove Mountain area) and Meredith outbreaks were
completely sequenced. Some variant viruses that occurred during the outbreaks
of NDV in these areas were also completely sequenced and the biological
properties of these isolates investigated in poultry. In all, ten complete
genome sequences were determined and differences noted in the rate of mutation
for certain genes.
Molecular analyses based on the study of the genomic sequence(s) of circulating Australian and overseas NDVs showed that the virus that was the cause of the initial outbreak of Newcastle disease was related to a previously described avirulent NDV isolated from Peats Ridge in the Mangrove Mountain area in 1998, rather than the incursion of an exotic overseas virulent virus. The evolution of the virus responsible for the outbreak of virulent NDV in Australia (termed the ‘progenitor’ virus) was also characterised at the molecular level. From these studies it was shown that the ‘progenitor’ virus was slowly evolving, as determined from its nucleotide sequence at its initial isolation in 1998 to that seen in 2001. However, analyses of these genome sequences demonstrated that the ‘progenitor’ virus did not appear to have a fusion cleavage signal sequence, which determines the virulence of the virus, and was prone to greater genetic variation than any other region in its genome.
Comparison of the ‘progenitor’
virus with overseas and earlier Australian isolates
The ‘progenitor virus’ had
some unique characteristics which were to become important in subsequent
investigations. The nucleotide sequence of its fusion (F) gene was initially
observed to be only two nucleotides different from that of a virulent virus,
and the haemaglutinin-neuraminidase (HN) gene had an extension of nine
amino acids when compared to the normal amino acid sequence for this protein.
Comparison with Australian isolates and those from overseas showed it to
be related to Australian NDV isolates and not overseas isolates. In particular,
two isolates which were isolated in NSW in 1986 and in Queensland in 1987
were shown to have a genetic relationship with both the ‘progenitor’ virus
and with the virulent isolates made in NSW and Victoria (1998-2002). From
a retrospective survey of NDV isolates, this ‘ancestor’ virus to the ‘progenitor’
and virulent viruses was demonstrated to have been present at Mangrove
Mountain NSW in 1991.
Analysis of individual
virus sequences comprising field isolates (quasi-species analysis)
Gene sequence analysis of
the individual viruses present in a field isolate of NDV was done to investigate
the genetic variability present in these isolates. It was determined that,
for a defined region of 500 nucleotides over the fusion gene cleavage site,
approximately 50% of the individual viruses that made up the field isolate
had the same nucleotide sequence as that determined to be the sequence
of the field isolate. However, the remaining individual viruses contained
mutations. These mutations occurred randomly in either the first, second
or third nucleotide of a codon for an amino acid. The latter usually did
not result in the alteration of the amino acid sequence of the protein,
however the other two mutation positions generally resulted in the generation
of an amino acid change. These amino acid changes were predominantly conservative
substitutions; however some radical changes were observed.
Gene sequence analysis showed that some field isolates of NDV with a relatively high intracerebral pathogenicity index (ICPI) had an avirulent F cleavage site. Quasi-species analysis of these field isolates showed that virulent virus was present up to a level of 26% of the viral population. However, poultry infected with these isolates behaved as if they were infected with an avirulent NDV. Realtime PCR demonstrated that, in some isolates of progenitor virus, virulent NDV sequences were present at a low level of 1:1,000 to 1:5,000.
Experiments which analysed the error rate as well as the total variants within a quasi-species showed that only two to three rounds of replication, in vivo or in vitro, are necessary for a plaque-purified virus to approximate the level of variation of the viral population seen in field isolates.
Detection of virulent
virus in field isolates using tissue culture breakthrough
Plaque-purified avirulent
NDV grown in tissue culture demonstrated the emergence of virulent virus
by mutation after a period of six to seven days. The level of virulent
virus that emerged after tissue culture was shown to vary from 1-18%. Virulent
virus was also demonstrated to be present in field isolates of NDV isolated
in Australia in the 1980s which belonged to a separate clade from those
associated with the 1998-2002 NDV outbreaks. These latter viruses where
shown to have HN extensions of seven amino acids. The presence of these
virulent viruses in an avirulent background remains undetected until a
selection pressure is applied.
Association of HN amino
acid extension with respiratory disease in poultry
Sequence analysis of a number
of overseas NDV isolates has shown that the HN protein is normally 571
amino acids in length; however, Australian NDV isolates have an additional
45 amino acids at the C-terminus of the HN protein. Sequence analyses of
many Australian isolates as part of this study showed that the C-terminal
extensions of the HN gene can vary from 7-45 amino acids. Australian NDV
isolates with HN extensions of 45 amino acids generally replicate in the
gut of the bird while those with HN extensions of 14, 9 and 7 amino acids
have been associated with “late summer respiratory disease” syndrome and
virus replication in the lungs and respiratory tract. The viruses generally
associated with this syndrome at present in Australia have 9 or 14 amino
acid extensions to their HN proteins.
Factors affecting the
emergence of virulent virus from an avirulent inoculum in poultry
Experiments were undertaken
in vivo to determine if the presence of a pre-existing avian pathogen could
influence the emergence of virulent ND virus from an avirulent virus. Preliminary
trials have shown that the presence of the coronavirus, infectious bronchitis
virus (IBV), during an NDV infection does not alter the apparent symptoms
nor virulence of NDV during infection. However the infection of birds with
infectious bursal disease virus (IBDV) prior to NDV infection may exacerbate
NDV disease symptoms, as well as potentially allowing virulent virus, which
has arisen by spontaneous mutation(s), to dominate the quasi-species population
replicating in poultry.
Conclusions and final
recommendations
Australia has currently
circulating a wide spectrum of NDV isolates that have now been well characterised
at the molecular level. Some well-defined molecular properties have been
ascribed to loci within the genome, the most important of these being the
virulence sequence at the fusion protein cleavage site. Another is the
possible link between the HN amino acid extension and viral replication
in the respiratory tract.
Over the period of this project, virulent NDV continued to emerge from an avirulent NDV background despite the implementation of quarantine control measures. Although V4 has proved effective as a vaccine, the temporal and geographic utilisation of V4 may need to be co-ordinated with a screening program to detect and eliminate the presence of the ‘progenitor’ virus.
The emergence of virulent virus in Australia is well connected to the presence of an avirulent ‘progenitor’ virus that, through natural mutation processes, can harbour virulent virus within its quasi-species. This virulent virus will not emerge from the avirulent background unless a selection pressure is placed on the quasi-species.
Another clade of NDV has been described which is also two nucleotides from virulence and has the same potential to initiate virulent NDV outbreaks in Australia. This clade is characterised by having a 7 amino acid HN extension. Virulent virus has been isolated from these field isolates.
It is recommended that a detailed survey be conducted within Australia to detect the presence of the ‘progenitor’ virus and eradicate it before the virus is spread further and causes further outbreaks. The role of other viral and environmental factors in the emergence of virulent virus from the ‘progenitor’ virus also needs to be further evaluated.
 
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