Defective interfering particles of mouse hepatitis virus

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.