Antigenic and phylogenetic studies on a variant Newcastle disease virus using anti-fusion protein monoclonal antibodies and partial sequencing of the fusion protein gene.

A virulent Newcastle disease virus (NDV) isolate, 34/90, reported to show considerable antigenic diversity from more classical strains of NDV, including vaccine strains, was evaluated phylogenetically and for the presence of neutralizing epitopes on the fusion protein. Comparison of a 309 nucleotide sequence of the fusion protein gene of 34/90 with other viruses confirmed the diversity of this virus, placing it in a discrete fifth genetic lineage with an avirulent virus isolated from waterfowl and genetically quite distant from other strains and isolates. The virus-neutralizing mAbs used in the present study were directed against at least seven distinct epitopes on the fusion protein. Of these seven, five are shared by 34/90 and the live vaccine virus Hitchner B1 and these plus an additional epitope are shared by 34/90 and strain Ulster 2C, which is used in inactivated vaccines. Two potential distinct epitopes were also shared by these three viruses. The results suggest that despite the detected antigenic and genetic variation of 34/90, it is unlikely that mutants which fail to be neutralized by antibodies induced by conventional vaccines would arise readily.

The nucleotide sequence of the HA1 of the haemagglutinin of an HI avian influenza virus isolate from turkeys in Germany provides additional evidence suggesting recent transmission from pigs.

The nucleotide sequence encoding the HA1 portion of the haemagglutinin gene of the influenza virus A/turkey/Germany/2482/90j isolated from birds kept in an area of many pig farms, was determined and compared with those of recent avian and swine influenza isolates. It was found to be closest to the 'avian-like' swine H1N1 influenza viruses that have been reported in Europe since the early 1980s and may represent good evidence for transmission of these viruses back to birds after they have become established in pigs.

Antigenic and genetic analyses of H1N1 influenza A viruses from European pigs.

H1N1 influenza A viruses isolated from pigs in Europe since 1981 were examined both antigenically and genetically and compared with H1N1 viruses from other sources. H1N1 viruses from pigs and birds could be divided into three groups: avian, classical swine and 'avian-like' swine viruses. Low or no reactivity of 'avian-like' swine viruses in HI tests with monoclonal antibodies raised against classical swine viruses was associated with amino acid substitutions within antigenic sites of the haemagglutinin (HA). Phylogenetic analysis of the HA gene revealed that classical swine viruses from European pigs are most similar to each other and are closely related to North American swine strains, whilst the 'avian-like' swine viruses cluster with avian viruses. 'Avian-like' viruses introduced into pigs in the UK in 1992 apparently originated directly from strains in pigs in continental Europe at that time. The HA genes of the swine viruses examined had undergone limited variation in antigenic sites and also contained fewer potential glycosylation sites compared to human H1N1 viruses. The HA exhibited antigenic drift which was more marked in 'avian-like' swine viruses than in classical swine strains. Genetic analyses of two recent 'avian-like' swine viruses indicated that all the RNA segments are related most closely to those of avian influenza A viruses.

An avian influenza virus of H10 subtype that is highly pathogenic for chickens, but lacks multiple basic amino acids at the haemagglutinin cleavage site.

Avian influenza virus isolate A/mandarin duck/Singapore/805/F-72/7/93 was found to be consistently highly pathogenic by recognised in vivo testing procedures although it was of a subtype (H10) not usually associated with high pathogenicity. The virus was also not typical of highly pathogenic influenza viruses in that it was not pathogenic when administered intra-nasally, did not possess a haemagglutinin cleavage site with multiple basic amino acids and did not replicate in the brains of chickens after intravenous inoculation. A re-examination of the earlier H10 isolate A/turkey/England/384/79 suggested that it was similarly pathogenic. The pathogenicity may have been associated with replication in the kidney.

Comparison of Newcastle disease viruses isolated from cormorants in Canada and the USA in 1975, 1990 and 1992.

Seventeen Newcastle disease virus (NDV) isolates obtained from cormorants, turkeys, a pelican, and a gull in Canada and the USA collected in 1975, 1990 and 1992 were analyzed for relatedness by monoclonal antibody profiling. In addition, nucleotide sequence analysis was performed in two areas of the fusion (F) gene for 5 of the isolates. No difference in the antigenicity of these 17 viruses, as determined by monoclonal antibody binding patterns, was seen. The amino acid sequences obtained via nucleotide sequencing at the cleavage site of the F protein showed that all the isolates tested had two pairs of basic amino acids immediately upstream of the cleavage site, and a phenylalanine residue at the N-terminus of the F1 protein, which is consistent with velogenic NDV. The deduced amino acid sequence obtained at the cleavage site of the F protein from 6 of the isolates was virtually identical regardless of the species, year of isolation, or location. However, the 1975 cormorant isolate showed marked differences from the 1990-1992 isolates in the nucleotide and deduced amino acid sequence of the F gene signal region. These data indicate that the 1990 and 1992 outbreaks were caused by the same epizootic virus and further suggest that the population of NDV in these wild birds may be very stable. The belief that the velogenic NDV circulating in cormorants in 1992 was transmitted into the free-ranging turkey flocks located near the cormorants in North Dakota is supported by the present study in which no distinction could be made between the viruses isolated from turkeys or wild birds.

Pathogenicity and phylogenetic evaluation of the variant Newcastle disease viruses termed "pigeon PMV-1 viruses" based on the nucleotide sequence of the fusion protein gene.

The nucleotide sequences of the entire F genes of two isolates of the pigeon PMV-1 (PPMV-1) variant of Newcastle disease virus (NDV) were determined using RTPCR. The deduced amino acid sequences of the F0 protein showed four differences between isolate 760/83 which had been passaged 4 times in chickens and gave an intravenous pathogenicity index in chickens (IVPI) of 2.01 and isolate 1168/84 which had received six passages in chickens and had an IVPI of 0.00. The F genes of virus from two passage levels of isolate 1447/84, 0 with IVPI value 0.00 and six with IVPI value 0.58, were partially sequenced to cover the areas of variation between 760/83 and 1168/84. The two passage levels of 1447/84 showed identical sequences in these areas which in turn were identical of those of 760/83. It was concluded that the recorded differences in intravenous pathogenicity were unlikely to be associated with differences in the primary structure of the F0 protein. Phylogenetic comparisons of the F gene sequences of the two PPMV-1 viruses with those published for other NDV strains and isolates showed that the PPMV-1 viruses formed a new fourth lineage but were closely related to strain Warwick with which they presumably shared a common origin.

Evaluation of the molecular basis of pathogenicity of the variant Newcastle disease viruses termed "pigeon PMV-1 viruses".

The amino acid sequence at the F2/F1 cleavage site was determined for 15 strains of the so-called pigeon PMV-1 (PPMV-1) variant of Newcastle disease virus (NDV) which showed close antigenic identity, determined by their reactions with a panel of 28 monoclonal antibodies, but considerable variation in their pathogenicity for chickens. Thirteen of the isolates possessed the motif 112G-R-Q-K-R-F117. This motif was seen for one virus which had initially low pathogenicity and remained unaltered when virulence of the virus for chickens was increased by bird to bird passage. The two other viruses had the sequence 112R-R-Q-K-R-F117 at the cleavage site which is more typical of virulent viruses, however, pathogenicity index tests indicated that these isolates were of moderate and low pathogenicity. The nucleotide sequence coding for the HN/HN0 extension region was determined for two of the PPMV-1 isolates. In both cases a stop codon was present indicating that the product for these viruses would be HN571. We conclude that the wide variation in pathogenicity of the variant PPMV-1 for chickens is not related to variation in the amino acid motif at the F2/F1 cleavage site nor due to production of HN0 which may also influence pathogenicity. The high virulence of some of the viruses examined confirms that a double pair of basic amino acids in the region of the F2/F1 cleavage site is not necessary for the full expression of virulence.

Head injuries at an inner city accident and emergency department.

A prospective study was carried out of 784 adults (aged 13 years and over) with recent head injuries who attended the Accident and Emergency Department of the Glasgow Royal Infirmary during an 11-week period (April-June 1978). One-third were caused by assault and only one-tenth by road traffic accidents. Half the patients had recently ingested alcohol, one-quarter of all patients had at least a brief period of post-traumatic amnesia (PTA). The overall admission rate was 28 per cent. Radiography of the skull was performed in 65 per cent and a fracture seen in 5 per cent of these. One-quarter of the patients had at least one unsatisfactory radiograph. Two patients whose fractures were initially missed on radiography were not admitted. Seven of the 24 patients with fractures had no clinical evidence of brain damage (no PTA, no impaired conscious level, no focal neurological signs or symptoms), but all had wounds of the scalp. One-third of all patients did not have radiography of the skull performed, nor were they admitted to hospital, yet one-quarter of these had some evidence of brain damage.