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Updated: Oct 25, 2025
Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses
Published on: April 4, 2019
Mutation Profiles of eGFP-Tagged Small Ruminant Morbillivirus During 45 Serial Passages in Ribavirin-Treated Cells
Fuxiao Liu1, Yanli Zou2, Lin Li2
1College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, China.
Abstract:
Small ruminant morbillivirus (SRMV), formerly known as peste-des-petits-ruminants virus, classified into the genus Morbillivirus in the family Paramyxoviridae. Its L protein functions as the RNA-dependent RNA polymerases (RdRp) during viral replication. Due to the absence of efficient proofreading activity in their RdRps, various RNA viruses reveal high mutation frequencies, making them evolve rapidly during serial passages in cells, especially treated with a certain mutagen, like ribavirin. We have previously rescued a recombinant enhanced green fluorescence protein-tagged SRMV (rSRMV-eGFP) using reverse genetics. In this study, the rSRMV-eGFP was subjected to serial passages in ribavirin-treated cells. Due to the ribavirin-exerted selective pressure, it was speculated that viral progenies would form quasispecies after dozens of passages. Viral progenies at passage-10, -20, -30, -40, and -50 were separately subjected to next-generation sequencing (NGS), consequently revealing a total of 34 single-nucleotide variations, including five synonymous, 21 missense, and one non-sense mutations. The L sequence was found to harbor eight missense mutations during serial passaging. It was speculated that at least one high-fidelity variant was present in viral quasispecies at passage-50. If purified from the population of viral progenies, this putative variant would contribute to clarifying a molecular mechanism in viral high-fidelity replication in vitro.
Insights
Small ruminant morbillivirus (SRMV) evolved under ribavirin pressure, generating quasispecies with numerous mutations. A potential high-fidelity variant emerged after 50 passages, offering insights into viral replication mechanisms.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Small ruminant morbillivirus (SRMV), a member of the Morbillivirus genus, utilizes an L protein functioning as an RNA-dependent RNA polymerase (RdRp).
- RNA viruses, including SRMV, exhibit high mutation rates due to limited RdRp proofreading activity, leading to rapid evolution, especially under mutagenic selective pressures like ribavirin.
Purpose of the Study:
- To investigate the genetic evolution of SRMV under ribavirin-induced selective pressure.
- To identify and characterize mutations arising during serial passages of a recombinant SRMV (rSRMV-eGFP).
- To explore the potential emergence of high-fidelity viral variants within a viral quasispecies population.
Main Methods:
- Rescue of a recombinant enhanced green fluorescence protein-tagged SRMV (rSRMV-eGFP) using reverse genetics.
- Serial passaging of rSRMV-eGFP in cells treated with the mutagen ribavirin.
- Next-generation sequencing (NGS) of viral progenies at multiple passage points (10, 20, 30, 40, and 50).
Main Results:
- A total of 34 single-nucleotide variations were identified across the sequenced viral progenies.
- These variations included five synonymous, 21 missense, and one non-sense mutation.
- The L protein sequence accumulated eight missense mutations during serial passaging, suggesting potential functional alterations.
Conclusions:
- Ribavirin treatment induced the formation of SRMV quasispecies with significant genetic diversity.
- The study speculates the presence of at least one high-fidelity variant within the viral quasispecies population at passage 50.
- Purification of this putative variant could elucidate molecular mechanisms underlying high-fidelity viral replication in vitro.
