Defective interfering particles of human parainfluenza virus 3 (HPIV3) were generated and/or amplified by serial undiluted passage of the standard virus. Analysis of the progeny virus titer at each cell passage revealed a cyclic pattern of virus production. Viruses produced from serial passages 8 and 9 interfered with replication of the standard virus. When cells were mixedly infected with standard virus and virus from either serial passages 5 or 8, three subgenomic RNA species, in addition to the standard virus genomic RNA, were detected in the progeny virions. Northern blot analysis revealed that all three subgenomic RNAs contained sequences from the 5$\sp\prime$-end of the standard virus genome. The data indicates that 5$\sp\prime$ defective interfering (DI) particles were generated and/or amplified during serial undiluted passage of HPIV3. Two independent HPIV3 persistent infections of LLC-MK$\sb2$ cells were established. One persistently infected culture was established by propagating the few cells which survived from an acute standard virus infection. The other persistently infected culture was readily established with virus from serial undiluted passage 8. This suggests that the DI particles present in serial passage 8 virus protected the cells from standard virus destruction. The two persistently infected cultures were propagated for nearly three years and the virus released from both cell lines at all times examined was noncytocidal. Subgenomic RNAs (putative DI particle genomes) were detected in virions released from both persistently infected cultures at various cell passages. No apparent changes in the size of the viral proteins were observed throughout the persistent infections. The DI particle-size genomes and the noncytocidal mutant virus are both likely to be involved in the maintenance of the persistent state. The degree of genetic change undergone by the viral genome during long-term persistence was examined. Nucleotide sequence analysis of the M gene and flanking regions of virus recovered after 147 cell passages (29 months of persistence) revealed that the viral genome did not undergo extensive genetic change. In the M protein coding region, only one conservative amino acid change was observed indicating that M protein mutation is not likely to be involved in the maintenance of the persistent infections. In contrast, the genomic 3$\sp\prime$-termini of viruses recovered after 146 cell passages was found to be highly mutated. The mutations observed were either U to C or A to G changes. Such exclusive substitution of one type of nucleotide for another is unlikely to be due to intrinsic polymerase errors but to an extrinsic biased hypermutational activity. An RNA unwinding/modifying activity that could give rise to the biased hypermutations is discussed.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/5615 |
| Date | January 1990 |
| Creators | Murphy, Donald G. |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 213 p. |
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