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Defective interfering particles of mouse hepatitis virus
Abstract:
After six to eight serial undiluted passages of mouse hepatitis virus (JHM strain) in DBT cell culture, a decrease in the yield of infectious virus occurred, and with further passages fluctuating yields of infectious virus were observed. The serially passaged virus interfered with the multiplication of the standard JHM virus, but not with vesicular stomatitis virus. After sucrose equilibrium centrifugation of high passage virus, a single peak contained both infectious virus and interfering activity. This virus population resembled the original JHM virus in its structural proteins, but it contained an increased proportion of a protein with a molecular weight of 65 X 10(3). Genomic RNA from standard JHM virus contained a single species of RNA with a molecular weight of 5.4 X 10(6). After five undiluted passages, however, the virion population contained two RNA species with molecular weights of 5.4 X 10(6) and 5.2 X 10(6). RNase T1 resistant oligonucleotide finger-printing of these RNAs showed that the lower molecular weight RNA had lost several oligonucleotide spots that were present in the genomic RNA of the standard JHM virus. After several serial diluted passages of passage 10 virus, a single virus population was obtained which again had only standard virus RNA with a molecular weight of 5.4 X 10(6) and lacked interfering activity. These results indicated that defective interfering particles were generated by serial undiluted passages of JHM virus.
Insights
Serial undiluted passages of mouse hepatitis virus generated defective interfering particles. These particles interfered with standard JHM virus replication but were eliminated by diluted passages.
Area of Science:
- Virology
- Molecular Biology
- Cell Culture
Background:
- Mouse hepatitis virus (JHM strain) is a significant pathogen.
- Serial passaging in cell culture is a common method for virus propagation and study.
- Understanding virus-host interactions and viral evolution is crucial for disease control.
Purpose of the Study:
- To investigate the effects of serial undiluted passaging on mouse hepatitis virus (JHM strain).
- To characterize the biological and molecular changes in JHM virus after extensive passaging.
- To determine the nature of the observed interference with standard JHM virus replication.
Main Methods:
- Serial undiluted and diluted passaging of JHM virus in DBT cell culture.
- Infectivity assays to measure virus yield.
- Interference assays against standard JHM virus and vesicular stomatitis virus.
- Sucrose equilibrium density gradient centrifugation.
- Analysis of viral structural proteins using SDS-PAGE.
- RNA extraction and analysis of genomic RNA species.
- RNase T1 resistant oligonucleotide fingerprinting.
Main Results:
- Serial undiluted passages led to decreased infectious virus yield and fluctuating titers.
- Passaged virus exhibited interference with standard JHM virus but not vesicular stomatitis virus.
- High-passage virus contained both infectious virus and interfering activity, with altered protein composition (increased 65 kDa protein).
- Genomic RNA analysis revealed the emergence of a lower molecular weight RNA species (5.2 x 10^6) alongside the standard RNA (5.4 x 10^6).
- Oligonucleotide fingerprinting indicated genomic alterations in the lower molecular weight RNA.
- Diluted passaging of high-passage virus restored the standard JHM virus RNA and eliminated interfering activity.
Conclusions:
- Serial undiluted passaging of JHM virus in DBT cells generates defective interfering particles (DIPs).
- These DIPs possess interfering activity and altered molecular characteristics, including changes in protein and RNA composition.
- The generation of DIPs is reversible through diluted passaging, suggesting a mechanism for viral genome modulation.