Reverse genetics technology offers the possibility to study the influence of particular amino acids of infectious bursal disease virus (IBDV) on adaptation to tissue culture. Genomic segments A and B of the very virulent (vv) IBDV field strain UK661 were completely cloned and sequenced, and the strain was rescued from full-length cDNA copies of both segments (UK661rev). Using site-directed mutagenesis, alteration of a single amino acid in the segment A-encoded VP2 (A284T) resulted in a limited capacity of UK661 to replicate in tissue culture. Additional alteration of a second amino acid (Q253H) increased replication efficiency in tissue culture. The second mutant (UK661-Q253H-A284T) was used to infect chickens and results were compared with UK661 and UK661rev. Whereas UK661 and UK661rev induced 100 % morbidity and 50-80 % mortality, UK661-Q253H-A284T proved to be strikingly attenuated, producing neither morbidity nor mortality. Moreover, UK661-Q253H-A284T-infected animals were protected from challenge infection. Thus, alteration of two specific amino acids in the VP2 region of IBDV resulted in tissue culture adaptation and attenuation in chickens of vvIBDV. The data demonstrate that VP2 plays a decisive role in pathogenicity of IBDV.

The nucleoprotein (NP) of Newcastle disease virus (NDV) functions primarily to encapsidate the virus genome for the purpose of RNA transcription, replication, and packaging. This conserved multifunctional protein is also efficient in inducing NDV-specific antibody in chickens. Here, we localized a conserved B-cell immunodominant epitope (IDE) spanning residues 447 to 455 and successfully generated a recombinant NDV lacking the IDE by reverse genetics. Despite deletion of NP residues 443 to 460 encompassing the NP-IDE, the mutant NDV propagated in embryonated specific-pathogen-free chicken eggs to a level comparable to that of the parent virus. In addition, a B-cell epitope of the S2 glycoprotein of murine hepatitis virus (MHV) was inserted in-frame to replace the NP-IDE. Recombinant viruses properly expressing the introduced MHV epitope were successfully generated, demonstrating that the NP-IDE not only is dispensable for virus replication but also can be replaced by foreign sequences. Chickens immunized with the hybrid recombinants produced specific antibodies against the S2 glycoprotein of MHV and completely lacked antibodies directed against the NP-IDE. These marked-NDV recombinants, in conjunction with a diagnostic test, enable serological differentiation of vaccinated animals from infected animals and may be useful tools in ND eradication programs. The identification of a mutation-permissive region on the NP gene allows a rational approach to the insertion of protective epitopes and may be relevant for the design of NDV-based cross-protective marker vaccines.The negative-strand RNA virus genome of Newcastle disease virus (NDV) contains six genes encoding six major structural proteins: nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN), and RNA-dependent RNA polymerase (L). The RNA together with NP, P, and L proteins forms the ribonucleoprotein complex (RNP), which serves as a template for RNA synthesis (15). The NP together with the polymerase proteins, P and L, plays an eminent role in encapsidating the RNA. Moreover, NP regulates transcription and replication of the viral genome by interacting with P alone, with P and L, or with itself (NP-NP interaction). For Sendai paramyxovirus, it was shown that a conserved N-terminal region of NP was involved in NP-RNA and NP-NP interaction (5), whereas the carboxyterminal domain was shown to be required for template function (8). Most of the NP is thus absolutely essential for virus replication due to multifold engagement of NP in the assembly and biologic activity of the RNP. In addition, NPs of negativestrand RNA viruses are highly immunogenic in nature and have been used as antigens for diagnostic purposes, including

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Editing of P-gene mRNA of Newcastle disease virus (NDV) enables the formation of two additional proteins (V and W) by inserting one or two nontemplated G residues at a conserved editing site (5-AAAAAGGG). The V protein of NDV plays an important role in virus replication and is also a virulence factor presumably due to its ability to counteract the antiviral effects of interferon. A recombinant virus possessing a nucleotide substitution within the A-stretch (5-AAgAAGGG) produced 20-fold-less V protein and, in consequence, was impaired in replication capacity and completely attenuated in pathogenicity for chicken embryos. However, in a total of seven serial passages, restoration of replication and pathogenic capacity in 9-to 11-day-old chicken embryos was noticed. Determining the sequence around the editing site of the virus at passage 7 revealed a C-to-U mutation at the second nucleotide immediately upstream of the 5-A 5 stretch (5-GuUAAgAAGGG). The V mRNA increased from an undetectable level at passage 5 to ca. 1 and 5% at passages 6 and 7, respectively. In addition, similar defects in another mutant possessing a different substitution mutation (5-AAAcAGGG) were restored in an identical manner within a total of seven serial passages. Introduction of the above C-to-U mutation into the parent virus (5-GuUAAAAAGGG) altered the frequency of P, V, and W mRNAs from 68, 28, and 4% to 15, 44, and 41%, respectively, demonstrating that the U at this position is a key determinant in modulating P-gene mRNA editing. The results indicate that this second-site mutation is required to compensate for the drop in edited mRNAs and consequently to restore the replication capacity, as well as the pathogenic potential, of editing-defective NDV recombinants. Newcastle disease virus (NDV) belongs to the new genusAvulavirus within the family Paramyxoviridae. NDV is the causative agent of one of the most devastating diseases of poultry worldwide. The negative-strand RNA virus genome of NDV encodes six genes (in the order 3Ј-NP-P-M-F-HN-L-5Ј). Except for the P gene mRNA, all express one major viral structural protein from a single open reading frame (ORF). The P gene mRNAs of the subfamily Paramyxovirinae express several proteins by the use of alternative reading frames and by a process of mRNA editing. The mRNA editing, which involves insertion of pseudotemplated G residue(s) occurs by a polymerase stuttering mechanism (10,11,24). Like in other members of paramyxovirinae, the P gene mRNA of NDV is edited by inserting one or two G residues into a G run within a conserved editing locus (5Ј-AAAAAGGG) (16, 21). As a result, three P gene derived mRNA species are produced which encode for the P, V, and W ORFs (unedited, with ϩ1 and ϩ2 frameshifts, respectively). The V and W proteins are colinear to the N-terminal half of the P protein but have different carboxy-terminal parts.The majority of the accessory proteins, including V and/or C proteins encoded by the P genes of different members of the subfamily Paramyxovirinae have been shown to be d...