Chad M. Fuller

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.

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).

Development of a real-time reverse-transcription PCR for the detection and simultaneous pathotyping of Newcastle disease virus isolates using a novel probe.

A real-time reverse-transcription PCR (RRT-PCR) was developed to detect and pathotype avian paramyxovirus type 1 (APMV-1), also known as Newcastle disease virus (NDV), which had been grown in embryonated fowls’ eggs. Two pairs of probes, VRP1 with ARP1 and VRP2 with ARP2, each with either the ‘universal base’ 2′ deoxyinosine incorporated or both inosines and locked nucleic acids (LNAs) incorporated, were designed to detect, respectively, a diverse range of virulent and avirulent viral templates that included the region coding for the fusion protein cleavage site. Oligonucleotide VRP1 hybridised with 76 of the 84 virulent isolates tested, while VRP2 detected 82, including 17 isolates with five or six template-probe mismatches. An alternative conventional probe, VRP3, with no inosine bases or LNAs, failed to hybridise 7 of 13 isolates, all of which tested positive with VRP2. Real-time assays with ARP1 showed that it detected 21 of the 28 avirulent isolates tested, and ARP2 detected 22/28, including one present in a mixture with virulent NDV. Neither probe was able to detect those isolates that were classified in genogroup six. All probes were specific for detecting either virulent or avirulent NDV. A specific PCR fragment of the predicted size was obtained, using the primer set designed for this study, with the 112 NDV isolates tested, including those in genogroup six. This assay demonstrates a rapid means for simultaneous detection and pathotyping of notifiable avian disease due to NDV.

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.

Partial characterisation of five cloned viruses differing in pathogenicity, obtained from a single isolate of pigeon paramyxovirus type 1 (PPMV-1) following passage in fowls' eggs

SummaryViruses with intracerebral pathogenicity indices (ICPIs) of 0.025, 0.55, 1.013 and 1.3. were cloned from a PPMV-1 isolate with an ICPI of 0.32 by passage in embryonated fowls’ eggs. Deduced amino acid sequences of the haemagglutinin-neuraminidase (HN) and precursor fusion proteins (F0) showed them to have only a single amino acid difference: those with an ICPI value <0.7 had proline at amino acid position 453 of the F0 protein, and those with an ICPI value >0.7 contained a serine. The virus with an ICPI of 0.025 was further passaged, and the ICPI of non-cloned virus increased to 0.76/0.79, which was then reduced to 0.49 on cloning. The proline at residue 453 was retained, but there were two nucleotide changes in the virus of ICPI 0.49, T → C at position 1769 in the untranslated region of the fusion gene and G → A at position 437 of the HN gene, resulting in the amino acid change G → R at position 116 in the HN protein.

Phylogenetic analysis of the nucleotide sequences for the HN gene of 22 avian paramyxovirus type 2 viruses reveals marked heterogeneity.

The nucleotide sequence of the HN gene was determined for 21 isolates of avian paramyxovirus type 2 virus and compared with the published HN gene of APMV-2/chicken/California/Yucaipa/56. The HN gene of the 22 viruses had five different lengths in the range of 1737 to 1755 nucleotides coding for 579 to 585 amino acids. Phylogenetic analysis of a corresponding 1734-nucleotide sequence from the HN gene of each virus established five genetic groups (I to V), two of which (II and IV) could be divided into two sub-groups (IIa and IIb; and IVa and IVb). Although there were some exceptions, generally isolates placed in the same genetic group had >80% similarity in nucleotide sequence and <80% with the other isolates; while those in the same sub-group had >90% nucleotide sequence similarity.

Inactivation of the infectivity of two highly pathogenic avian influenza viruses and a virulent Newcastle disease virus by ultraviolet radiation

Exposure of a virulent isolate of Newcastle disease virus (NDV) and two highly pathogenic avian influenza (HPAI) viruses, one of H7N1 subtype and the other H5N1 subtype, to a continuous ultraviolet B flux of approximately 90µW/cm2, which models solar ultraviolet radiation, resulted in an exponential decline in infectivity with time. The time taken for a reduction in titre of 1 log10 median tissue culture infectious dose for each virus was: NDV, 69 min; H7N1 HPAI virus, 158 min; and H5N1 HPAI, virus 167 min.

Newcastle disease: a continuing threat to UK poultry

AT the end of 2010 and beginning of 2011 several Newcastle disease outbreaks in unvaccinated poultry associated with pigeon paramyxovirus type 1 (PPMV-1) infection were officially reported by two northern European countries (OIE 2010, 2011a, b, c). Infected wild birds were suspected to be the source of the virus, presumably coming into contact with poultry, or gaining access to poultry houses, during the severe winter conditions. PPMV-1 is a virulent ‘pigeon variant’ Newcastle disease virus (of avian paramyxovirus genotype 4b/VIb) and the cause of the continuing panzootic that began in racing and feral pigeons more than 30 years ago. PPMV-1 strains are also fully capable of infecting …