Canine distemper virus nucleocapsid: Relevance to persistence

In order to monitor altered patterns of N gene expression within canine distemper virus (CDV) infected cells, probes complementary to N gene sequences and hybridization assays for their employment were developed. A cDNA library was prepared from CDV messenger RNA and an insert complementary to the N gene identified. A dot-blot hybridization procedure for the detection and quantitation of viral nucleic acids within lytically or persistently infected cells was established. The sensitivity of the procedure exceeded that of immunofluorescence assays. In addition, an assay was established allowing the detection of lytic and persistent virus sequences in situ.; N and P protein monospecific antisera were generated to examine translational levels of specific virus core protein. A procedure was developed permitting the efficient recovery of undegraded N and P antigen for the production of monospecific hyperimmune serum. Antisera were capable of detecting viral antigen within CDV lytically and persistently infected cells by immunofluorescence. The NC isolation procedure also made possible recovery of intact genomic RNA and the ultrastructural evaluation of NC variants.; Using this procedure, three NC variants were defined: light-NC (L-NC), dense-NC (D-NC), and defective-NC (Df-NC). Untrastructural differences between L-NC and D-NC correlated to the incorporation of a 70 kdal non-viral protein into the basic NC structure of L-NC. Df-NC encapsidated subgenomic RNAs with lengths corresponding to paramyxoviral defective interfering genomes.; The identification of determinants affecting the association of the 70 kdal nucleocapsid associated protein (NAP) with the virus NC were also pursued. Vero cell lines were identified exhibiting a gradation of NAP production corresponding to L-NC levels produced upon infection. Plaque size was proportional to levels of NAP, supporting the contention that L-NC represents a more biologically active variant. It was shown that the high pass cells supported the lowest levels of L-NC, with increases effected by heat shock. High and low NAP binding virus pools were also identified, demonstrating that virus phenotype also determines NAP-NC association. Based on these observations, hypotheses relative to age related susceptibility to virus disease, evolution of persistent from lytic virus infection, and virus tissue tropisms were formulated.