In the last decade many studies have been performed on the virulence of Newcastle disease virus (NDV). This is mainly due to the development of reverse genetics systems which made it possible to genetically modify NDV and to investigate the contribution of individual genes and genome regions to its virulence. However, the available information is scattered and a comprehensive overview of the factors and conditions determining NDV virulence is lacking. This review summarises, compares and discusses the available literature and shows that virulence of NDV is a complex trait determined by multiple genetic factors.

The generation of deletion mutants, including defective interfering viruses, upon serial passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in insect cell culture has been studied. Sequences containing the non-homologous region origin of DNA replication (non-hr ori) became hypermolar in intracellular viral DNA within 10 passages in Se301 insect cells, concurrent with a dramatic drop in budded virus and polyhedron production. These predominant non-hr ori-containing sequences accumulated in larger concatenated forms and were generated de novo as demonstrated by their appearance and accumulation upon infection with a genetically homogenous bacterial clone of SeMNPV (bacmid). Sequences were identified at the junctions of the non-hr ori units within the concatemers, which may be potentially involved in recombination events. Deletion of the SeMNPV non-hr ori using RecE/RecT-mediated homologous ET recombination in Escherichia coli resulted in a recombinant bacmid with strongly enhanced stability of virus and polyhedron production upon serial passage in insect cells. This suggests that the accumulation of non-hr oris upon passage is due to the replication advantage of these sequences. The non-hr ori deletion mutant SeMNPV bacmid can be exploited as a stable eukaryotic heterologous protein expression vector in insect cells.Baculoviruses are large enveloped, circular double-stranded DNA insect viruses which are widely used as bioinsecticides in agriculture and forestry and can be genetically engineered to improve their effectiveness (2, 18). More recently, baculoviruses were shown to have potential as gene delivery vectors for gene therapy (12,32,45) or as vectors for surface display of complex eukaryotic proteins (6). Yet, their major application to date is as a viral vector for the expression of heterologous proteins in insect cells (19,36). The prototypic and most intensively studied baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), has been primarily used as an expression vector, while other baculoviruses may become exploited as well, especially when appropriate cell lines are available.A major drawback in the large-scale production of baculoviruses as bioinsecticides or for heterologous protein production is the so-called passage effect. This effect is notable as a significant drop in production after prolonged virus passaging in insect cell culture (reviewed by Krell [25]) and is a result of the accumulation of defective interfering particles (DIs) (20). These DIs are rapidly generated in cell culture (39) and become predominant after prolonged passaging, meanwhile interfering with the replication of intact helper virus. However, the mechanism of the generation of DIs is still enigmatic, and the sequences involved are unknown.DIs have retained cis-acting elements essential for baculovirus DNA synthesis, such as origins of replication (ori) (25). Transient virus-mediated plasmid replication assays demonstrated that baculovirus homologous regions (hrs) (21,22,28,37), as...

Virulent strains of Newcastle disease virus ([NDV]also known as avian paramyxovirus type 1) can be discriminated from low-virulence strains by the presence of multiple basic amino acid residues at the proteolytic cleavage site of the fusion (F) protein. However, some NDV variants isolated from pigeons (pigeon paramyxovirus type 1 [PPMV-1]) have low levels of virulence, despite the fact that their F protein cleavage sites contain a multibasic amino acid sequence and have the same functionality as that of virulent strains. To determine the molecular basis of this discrepancy, we examined the role of the internal proteins in NDV virulence. Using reverse genetics, the genes encoding the nucleoprotein (NP), phosphoprotein (P), matrix protein (M), and large polymerase protein (L) were exchanged between the nonvirulent PPMV-1 strain AV324 and the highly virulent NDV strain Herts. Recombinant viruses were evaluated for their pathogenicities and replication levels in day-old chickens, and viral genome replication and plaque sizes were examined in cell culture monolayers. We also tested the contributions of the individual NP, P, and L proteins to the activity of the viral replication complex in an in vitro replication assay. The results showed that the replication proteins of Herts are more active than those of AV324 and that the activity of the viral replication complex is directly related to virulence. Although the M protein affected viral replication in vitro, it had only a minor effect on virulence.Newcastle disease is a severe infectious disease of birds caused by Newcastle disease virus (NDV), or avian paramyxovirus type 1 (APMV-1). NDV is classified in the genus Avulavirus of the family Paramyxoviridae (32) and has a singlestranded, negative-sense RNA genome consisting of six genes in the order 3Ј-NP-P-M-F-HN-L-5Ј (28) that encode at least seven proteins: the nucleocapsid protein (NP), the phosphoprotein (P), the matrix protein (M), the fusion protein (F), the hemagglutinin-neuraminidase protein (HN), and the polymerase protein (L). During P gene transcription, an additional, nonstructural protein (V) is produced by means of mRNA editing (55).The M, F, and HN proteins are associated with the viral envelope. The F and HN proteins mediate entry and release, and the M protein is involved in the morphogenesis and budding of NDV (28). The V protein is involved in interferon antagonism (42). The NP protein encapsidates the RNA genome to form the nucleocapsid, which serves as the template for viral transcription and replication. The P protein is essential for viral RNA synthesis and has multiple roles (10, 12). It forms separate complexes with the NP and L proteins and the nucleocapsid (22). Transcription of the viral genomic RNA occurs by way of the viral polymerase (P-L complex); the catalytic activities of the polymerase are functions of the L protein, and the P protein is responsible for the binding of the P-L complex to the nucleocapsid. Once sufficient viral proteins are generated, NP starts to bind to the leader ch...