Mutation Profiles of eGFP-Tagged Small Ruminant Morbillivirus During 45 Serial Passages in Ribavirin-Treated Cells.

Fuxiao Liu,Chunju Liu,Yanli Zou,Lin Li,Xiaodong Wu

doi:10.3389/fvets.2021.690204

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.

Highlights

Peste des petits ruminants is a highly contagious disease, mainly affecting goats and sheep, and even large ruminants [1,2,3]
The P5 rSRMV-Enhanced green fluorescence protein (eGFP) was serially passaged in ribavirin-treated VDS cells
The eGFP facilitated observation of fluorescent syncytium formation on a cell monolayer in real time. Such a feature prompted us to use the rSRMV-eGFP for uncovering viral mutation profiles during serial dozens of passages

Summary

Introduction

Peste des petits ruminants is a highly contagious disease, mainly affecting goats and sheep, and even large ruminants [1,2,3]. Its etiological agent is small ruminant morbillivirus (SRMV), formerly known as peste-des-petits-ruminants virus. Its genome is a single strand of RNA with negative polarity [4], coding for six structural proteins, namely nucleocapsid (N), phospho (P), matrix (M), fusion (F), hemagglutinin (H), and large (L) proteins in the order of 3′-N-P (V/C)-M-F-H-L-5′. Viral RNA genome is encapsulated by the N protein forming a helical nucleocapsid, in combination with the L and P proteins to form a ribonucleoprotein complex [6]. It is assumed to carry all activities necessary for genomic RNA transcription and replication [5]

Methods

Results

Conclusion