No abstract

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.

A full-length cDNA clone of Newcastle disease virus (NDV) vaccine strain LaSota was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Transfection of this plasmid into cells that were infected with a recombinant fowlpoxvirus that expressed T7 RNA polymerase, resulted in the synthesis of antigenomic NDV RNA. This RNA was replicated and transcribed by the viral NP, P, and L proteins, which were expressed from cotransfected plasmids. After inoculation of the transfection supernatant into embryonated specific-pathogen-free eggs, infectious virus derived from the cloned cDNA was recovered. By introducing three nucleotide changes in the cDNA, we generated a genetically tagged derivative of the LaSota strain in which the amino acid sequence of the protease cleavage site (GGRQGR↓L) of the fusion protein F0 was changed to the consensus cleavage site of virulent NDV strains (GRRQRR↓F). Pathogenicity tests in day-old chickens showed that the strain derived from the unmodified cDNA was completely nonvirulent (intracerebral pathogenicity index [ICPI] = 0.00). However, the strain derived from the cDNA in which the protease cleavage site was modified showed a dramatic increase in virulence (ICPI = 1.28 out of a possible maximum of 2.0). Pulse-chase labeling of cells infected with the different strains followed by radioimmunoprecipitation of the F protein showed that the efficiency of cleavage of the F0 protein was greatly enhanced by the amino acid replacements. These results demonstrate that genetically modified NDV can be recovered from cloned cDNA and confirm the supposition that cleavage of the F0 protein is a key determinant in virulence of NDV.

We examined the influence of inactivation of various genes located in the unique short (Us) region of pseudorabies virus on virus replication and assembly in porcine nasal mucosa explant cultures. The following strains were used: the virulent wild-type strain NIA-3, and strains derived from NIA-3 containing a mutation inactivating the genes encoding either the US3-encoded protein kinase (PK), gG, gD, gI, gE, the 28 kDa (' 28K') protein (single mutant), or the 28K and 11 kDa ('11K') proteins (double mutant). In addition a wild-type rescuant was used, which was generated by marker rescue from a PK mutant. All virus strains infected nasal epithelium and had invaded the stroma after approximately 24 h. The morphogenesis in nasal epithelium cells of two PK-mutants showed the most striking differences compared to the parent NIA-3 strain and the other mutant strains. The changes could be ascribed to the US3-encoded PK because the rescue mutant showed a similar morphogenesis to wild-type NIA-3. The transmembrane transport of the PKmutants was impaired at the outer nuclear membrane which resulted in an accumulation of virions in the perinuclear space. These results suggest that proteins, phosphorylated by the US3-encoded PK, are involved in debudding of virus particles at the outer nuclear membrane. This defect in the transport of the US3 mutant probably explains their reduced replication in vitro. The gG-, gD , gI , gEL 28K-and 11K-mutant strains showed minor or no changes in viral assembly. Thus the reported decreased virulence of the gD , gF and gE mutants was, in contrast to that of the PKmutants, not associated with clear alterations in morphogenesis.

No abstract