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Newcastle disease virus (NDV) strains, isolated from outbreaks during epizootics between 1992 and 1996 in Western European countries, were compared by restriction enzyme cleavage site mapping of the fusion (F) protein gene between nucleotides 334 and 1682 and by sequence analysis between nucleotides 47 and 435. Both methods revealed that NDV strains responsible for these epizootics belong to two distinct genotypes. Strains derived from sporadic cases in Denmark, Sweden, Switzerland and Austria were classified into genotype VI [6], the same group which caused outbreaks in the Middle East and Greece in the late 1960's and in Hungary in the early 1980's. In contrast, viruses that caused epizootics in Germany, Belgium, The Netherlands, Spain and Italy could be classified into a novel genotype (provisionally termed VII), hitherto undetected in Europe. It is possible that the genotype VII viruses originated in the Far East because they showed a high genetic similarity (97%) to NDV strains isolated from Indonesia in the late 1980's.

34 strains of Newcastle disease virus (NDV) isolated during epizootics in the Republic of South Africa and in Mozambique between 1990 and 1995, and in Bulgaria and Turkey in 1995-1997 were identified by restriction enzyme and partial sequence analysis of the fusion (F) protein gene. The majority of isolates in southern Africa and those from Bulgaria and Turkey were placed into a novel group which has been termed VIIb. Group VIIb is part of a larger genetic cluster (VII) that also includes NDV strains from the Far East and some western European countries (VIIa). The genetic distance of 7-8, 5% between genotype VIIa and VIIb viruses excludes the existence of a direct epidemiological link between recent southern African epizootics and outbreaks in either western Europe in the 1990's or those of the Far East. Another hitherto unrecorded genotype (VIII) was also found in South Africa with descendants of putative ancestral members isolated in the 1960's. The genetic distance of recent group VIII strains from the major epizootic genotype (VIIb) is over 11%, therefore outbreaks caused by them were epidemiologically unrelated. Genotype VIII viruses must have been maintained in South Africa by endemic infections during the past decades while group VIIb appears to be introduced more recently.

A 75% region of the F gene (between nucleotides 334 and 1682) of Newcastle disease virus (NDV) RNA was amplified by reverse transcription polymerase chain reaction (RT-PCR). PCR products were cleaved by three restriction endonucleases and the positions of thirty cleavage sites were mapped in more than 200 NDV strains. Restrictions site analysis established six major groups of NDV isolates and unique fingerprints of vaccine strains. Group I comprised lentogenic strains isolated mainly from waterfowl with some from chickens. "Old" (prior to 1960s) North American isolates of varying virulence including lentogenic and mesogenic vaccine strains belonged to group II. Group III included two early isolates from the Far East. Early European strains (Herts 33 and Italien) of the first panzootic (starting in the late 1920s) and their descendants with some modifications were placed into group IV. NDV strains isolated during the second panzootic of chickens (starting in the early 1960s) were classified into two groups. Group V included strains originating in imported psittacines and in epizootics of chickens in the early 1970s. Group V1 comprised strains from the Middle East in the late 1960s and later isolates from Asia and Europe. Pigeon paramyxovirus-1 strains that were responsible for the third panzootic formed a distinct subgroup in group V1. Our grouping of NDV strains has confirmed group differences established by monoclonal antibodies. It is concluded that restriction site analysis of F gene PCR amplicons is a relatively fast, simple and reliable method for the differentiation and identification of NDV strains.

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Analysis of the live attenuated pseudorabies virus (PrV) vaccine strain Bartha indicated location of a major determinant for PrV neurovirulence within the genomic BamHI fragment 4 (B. Lomniczi et al., 1984, J. Virol. 52, 198-205). To more precisely localize the defect, marker rescue experiments were performed using cloned subfragments of BamHI-4. Rescuants were analyzed after intracerebral infection for their virulence in chicken, as well as after intranasal infection for virulence in pigs. We show that the defect associated with attenuation in strain Bartha is located in a 3.8-kb subfragment of BamHI-4 which encompasses the PrV UL20 and UL21 genes and a putative origin of replication (B. Klupp, H. Kern, and T. C. Mettenleiter, 1992, Virology 191, 900-908). Sequence analysis of this region of the strain Bartha genome and comparison with the corresponding region in wild-type PrV strain Ka revealed the presence of eight point mutations. Four nucleotide exchanges reside within the UL21 gene with three of them leading to amino acid substitutions; one is located in the intergenic region between the UL20 and UL21 genes and three are localized downstream from the UL21 gene. Neither the UL20 gene nor the putative origin sequence was affected. Insertional inactivation of the UL21 gene in wild-type PrV strain Ka led to a marked attenuation of the virus for pigs infected by the intranasal route. In summary, our data show that the PrV UL21 gene is a major determinant of PrV virulence and that point mutations affecting the UL21 gene of live vaccine strain Bartha contribute to its attenuated phenotype.