E. W. Aldous

A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene

A sequence 375 nucleotides in length, which included the region encoding the cleavage activation site and signal peptide of the fusion protein gene, was determined for 174 isolates of Newcastle disease virus (avianparamyxovirus type 1). These were compared with the sequences of 164 isolates published on GenBank, and the resulting alignment was analysed phylogenetically using maximum likelihood. The results are presented asunrooted phylogenetic trees. Briefly, the isolates divided into six broadly distinct groups (lineages 1 to 6).Lineages 3 and 4 were further subdivided into four sublineages (a to d) and lineage 5 into five lineages (a to e).Considerable genetic heterogeneity was detected within avian paramyxoviruses type 1, which appears to beinfluenced by host, time and geographical origin. It is concluded that by using this dataset it will be possible totype future virus isolates rapidly on the basis of their nucleotide sequence and make inferences about theirorigins.

The long view: a selective review of 40 years of Newcastle disease research

This review is written for the series celebrating the 40th year since the first issue of Avian Pathology. The aim of the authors was to cover the developments in Newcastle disease (ND) research over the last 40 years that they considered significant. During those 40 years there have been several panzootics of this serious disease in poultry and for the last 30 years there has been a continuing panzootic in domestic pigeons, which has spread to wild birds and poultry. The 40-year period began with worldwide outbreaks of severe ND, which served as an important impetus for ND research work. Although early work was concerned with controlling the disease, specifically by improving and developing new vaccines and vaccine regimens, even prior to the 1970s ND virus was seen as a useful laboratory virus for replication and virulence studies. This review covers the historical developments in the following areas: understanding the molecular basis of virulence; epidemiology and relatedness of different ND strains, both antigenically and genetically; the emergence of virulent strains and their relationship with viruses of low virulence; sequencing and understanding the viral genome and genes; the development of rapid molecular-based diagnostic tests; and the phylogeny and molecular taxonomy of ND virus. The authors suggest areas in which future research could or should be undertaken.

ANTIGENIC AND GENETIC CHARACTERISATION OF NEWCASTLE DISEASE VIRUSES ISOLATED FROM OUTBREAKS IN DOMESTIC FOWL AND TURKEYS IN GREAT BRITAIN DURING 1997

Antigenic and genetic analyses of viruses from the 11 outbreaks of Newcastle disease in Great Britain, 12 of the outbreaks in Northern Ireland and the single outbreak in the Republic of Ireland which occurred in 1997, indicated that they were all essentially similar. In addition, the viruses from the British Isles were very similar to viruses isolated from three outbreaks in pheasants in Denmark between August and November 1996, from a goosander in Finland in September 1996, from an outbreak in chickens in Norway in February 19,97, and from an outbreak in chickens in Sweden in November 1997. Viruses from outbreaks in other countries during 1995 to 1997 could be distinguished antigenically and/or genetically from the 1996 to 1997 Scandinavian/British Isles isolates, as could viruses responsible for two separate outbreaks in caged birds in quarantine premises in Great Britain in March 1997. Minor nucleotide differences in the 413-base region of the fusion gene and the 187-base region of the haemagglutinin-neuraminidase gene sequenced in this study allowed the 1996 to 1997 Scandinavian/British Isles isolates to be divided into groups. These groups broadly corresponded to the clusters of disease outbreaks, but suggested that the discrete outbreak in Scotland was probably the result of virus spread from Northem Ireland. Overall, the antigenic and genetic analyses of these viruses were consistent with the theory that the virus was introduced into the British Isles by migratory birds moving from north-east Europe. However, it was not possible to rule out other sources, such as the movement of pheasants from Denmark.

Rapid pathotyping of Newcastle disease virus (NDV) using fluorogenic probes in a PCR assay.

Hybridisation of PCR fragments with fluorogenic probes specific for pathotype allowed an estimation of pathogenicity of Newcastle disease virus (NDV) isolates using a modified TaqMan procedure. Six probes were used, designed to recognise nucleotide sequences in the fusion protein gene sequence corresponding to the precursor protein F0 cleavage site of both virulent and avirulent viruses. Forty-three of the 45 isolates tested, including 18 examined in a blind study were pathotyped successfully and rapidly, with close correlation between cleavage site nucleotide sequences, TaqMan results and intracerebral pathogenicity index (ICPI) values. One isolate, which could not be pathotyped by nucleotide sequencing, was shown using the TaqMan system to be a mixture of virulent and avirulent NDV. The results of this study suggest that using this modified TaqMan protocol, the likely virulence of most ND isolates can be determined rapidly and reproducibly.

Infection dynamics of highly pathogenic avian influenza and virulent avian paramyxovirus type 1 viruses in chickens, turkeys and ducks

A range of virus doses were used to infect 3-week-old chickens, turkeys and ducks intranasally/intraocularly, and infection was confirmed by the detection of virus shedding from the buccal or cloacal route by analysis of swabs collected using real-time reverse transcriptase-polymerase chain reaction assays. The median infectious dose (ID50) and the median lethal dose (LD50) values for two highly pathogenic avian influenza (HPAI) viruses of H5N1 and H7N1 subtypes and one virulent Newcastle disease virus (NDV) were determined for each virus and host combination. For both HPAI viruses, turkeys were >100-fold more susceptible to infection than chickens, while both these hosts were >10-fold more susceptible to H5N1 virus than the H7N1 virus. All infected chickens and turkeys died. Ducks were also much more readily infected with the H5N1 virus (ID50≤101 median embryo infective dose [EID50]) than the H7N1 virus (ID50=104.2 EID50). However, the most notable difference between the two viruses was their virulence for ducks, with a LD50 of 103 EID50 for the H5N1 virus, but no deaths in ducks being attributed to infection with H7N1 virus even at the highest dose (106 EID50). For both HPAI virus infections of ducks, the ID50 was lower than the LD50, indicating that infected birds were able to survive and thus excrete virus over a longer period than chickens and turkeys. The NDV strain used did not appear to establish infection in ducks even at the highest dose used (106 EID50). Some turkeys challenged with 106 EID50, but not other doses, of NDV excreted virus for a number of days (ID50=104.6 EID50), but none died. In marked contrast, chickens were shown to be extremely susceptible to infection and all infected chickens died (ID50/LD50=101.9 EID50).

Outbreak of Newcastle disease due to pigeon paramyxovirus type I in grey partridges (Perdix perdix) in Scotland in October 2006.

In October 2006, following an initially non-statutory disease investigation affecting 12-week-old grey partridges (Perdix perdix), an outbreak of Newcastle disease due to infection with the avian paramyxovirus type 1 virus responsible for the current panzootic in pigeons (PPMV-1) was confirmed in Scotland. Two pens of partridges were affected by signs including loss of condition, diarrhoea, progressive neurological signs and mortality totalling approximately 24 per cent, and laboratory evidence of the infection was obtained only in these groups. The premises had approximately 17,000 poultry including a collection of 375 birds of rare breeds, containing endangered breeds of significant conservation value, which were not culled but subjected to a health monitoring and testing programme. Investigations suggested that a population of feral pigeons living above the affected pens of partridges was the likely source of the outbreak. Laboratory and genetic analyses confirmed that the isolate recovered from the clinically affected partridges was PPMV-1, belonging to genetic lineage 4b. However, the virus could not be isolated from or detected in dead pigeons collected from the affected buildings.

Outbreak of Newcastle disease in pheasants (Phasianus colchicus) in south-east England in July 2005

E. W. Aldous, BSc, MSc, PhD, R. J. Manvell, CBiol, MIBiol, W. J. Cox, V. Ceeraz, BSc, W. Shell, BSc, MSc, D. J. Alexander, BTech, PhD, FIBiol, FRCPath, DSc, I. H. Brown, CBiol, MIBiol, PhD, Virology Department, Veterinary Laboratories Agency – Weybridge, New Haw, Addlestone, Surrey KT15 3NB D. G. Harwood, BVetMed, MRCVS, Veterinary Laboratories Agency – Winchester, Itchen Abbas, Winchester, Hampshire

Experimental assessment of the pathogenicity of the Newcastle disease viruses from outbreaks in Great Britain in 1997 for chickens and turkeys, and the protection afforded by vaccination.

The Newcastle disease virus isolated from healthy turkeys in outbreak GB 97/6 was used to challenge 4-week-old turkeys and chickens, which were either not vaccinated or had received a single dose of Hitchner B1 live vaccine 14 days earlier, by one of the intramuscular, intranasal or contact routes. Similar experiments were done in 38-day-old turkeys and chickens using virus isolated from severely sick chickens in outbreak GB 97/1. All vaccinated chickens showed low but measurable immune responses 14 days after vaccination, but only three of the turkeys had detectable antibodies. No vaccinated turkey or chicken showed any clinical sign after challenge with either virus. The virus from healthy turkeys in outbreak GB 97/6 induced clinical signs in 12/30 unvaccinated turkeys after challenge and 7/30 died. In unvaccinated chickens, challenge with this virus produced clinical signs in 25/30 birds and 21/30 died. In challenge experiments with the virus from outbreak GB 97/1 in chickens, 3/30 unvaccinated turkeys showed clinical signs and all three subsequently died. In contrast, 30/30 unvaccinated chickens challenged with this virus showed clinical signs and died. Vaccination did not prevent infection and excretion of either challenge virus. However, when compared with unvaccinated birds, vaccination reduced significantly the length of time virus was excreted and the overall proportion of swabs that were positive.

Two genetically closely related pigeon paramyxovirus type 1 (PPMV-1) variants with identical velogenic fusion protein cleavage sites but with strongly contrasting virulence

Two pathogenetically different pigeon paramyxovirus type 1 (PPMV-1) virus clones were recently derived by passage of a single isolate with an intracerebral pathogenicity index (ICPI) of 0.32. The virus clones had an ICPI of 0.025 and 1.3, respectively (Fuller et al., 2007). Remarkably both viruses contained a cleavage site motif in the precursor fusion (F) protein that is usually associated with virulent viruses. In the current study, both viral genomes were completely sequenced and only four amino acid differences were observed. Of these, two were considered irrelevant on theoretical grounds and two amino acid changes were unique for virus 0.025. The latter were introduced into an infectious clone of a virulent Newcastle disease virus strain, individually and combined, and the effects of the mutations on pathogenicity were examined. The results indicate that only the S453P substitution in the F protein had a modest effect on pathogenicity. We were not able to identify the molecular basis for the pathogenicity difference between both viruses. However, our observations emphasize the need to determine both the virulence (ICPI) and the sequence of the cleavage site of the F protein to avoid dismissing of potential virulent PPMV-1 isolates.

A molecular epidemiological investigation of avian paramyxovirus type 1 viruses isolated from game birds of the order Galliformes

The partial (370 nucleotides) fusion gene sequences of 55 avian paramyxovirus type 1 (APMV-1) isolates were obtained. Included were 41 published sequences, of which 16 were from strains of APMV-1 of previously determined lineages included as markers for the data analysed and 25 were from APMV-1 viruses isolated from game birds of the order Galliformes. In addition, we sequenced a further 14 game bird isolates obtained from the repository at the Veterinary Laboratories Agency. The game bird isolates had been obtained from 17 countries, and spanned four decades. Earlier studies have shown that class II APMV-1 viruses can be divided into at least 15 lineages and sub-lineages. Phylogenetic analysis revealed that the 39 game bird isolates were distributed across 12 of these sub-lineages. We conclude that no single lineage of Newcastle disease viruses appears to be prevalent in game birds, and the isolates obtained from these hosts reflected the prevailing, both geographically and temporally, viruses in poultry, pigeons or wild birds.