Rescuing eGFP-Tagged Canine Distemper Virus for 40 Serial Passages Separately in Ribavirin- and Non-Treated Cells: Comparative Analysis of Viral Mutation Profiles.

Fuxiao Liu,Jiahui Lin,Hu Shan,Qianqian Wang,Yilan Huang,Ning Wang,Youming Zhang

doi:10.3389/fcimb.2021.746926

Abstract

Due to lacking a proofreading mechanism in their RNA-dependent RNA polymerases (RdRp), RNA viruses generally possess high mutation frequencies, making them evolve rapidly to form viral quasispecies during serial passages in cells, especially treated with mutagens, like ribavirin. Canine distemper virus (CDV) belongs to the genus Morbillivirus. Its L protein functions as an RdRp during viral replication. In this study, a recombinant enhanced green fluorescence protein-tagged CDV (rCDV-eGFP) was rescued from its cDNA clone, followed by viral identification and characterization at passage-7 (P7). This recombinant was independently subjected to extra 40 serial passages (P8 to 47) in ribavirin- and non-treated cells. Two viral progenies, undergoing passages in ribavirin- and non-treated VDS cells, were named rCDV-eGFP-R and -N, respectively. Both progenies were simultaneously subjected to next-generation sequencing (NGS) at P47 for comparing their quasispecies diversities with each other. The rCDV-eGFP-R and -N showed 62 and 23 single-nucleotide mutations (SNMs) in individual antigenomes, respectively, suggesting that the ribavirin conferred a mutagenic effect on the rCDV-eGFP-R. The spectrum of 62 SNMs contained 26 missense and 36 silent mutations, and that of 23 SNMs was composed of 17 missense and 6 silent mutations. Neither the rCDV-eGFP-R nor -N exhibited nonsense mutation in individual antigenomes. We speculate that the rCDV-eGFP-R may contain at least one P47 sub-progeny characterized by high-fidelity replication in cells. If such a sub-progeny can be purified from the mutant swarm, its L protein would elucidate a molecular mechanism of CDV high-fidelity replication.

Highlights

Canine distemper is a severe infectious disease, affecting a broad variety of domestic and wild carnivores (McCarthy et al, 2007)
The rescued rCDV-enhanced green fluorescence protein (eGFP) was subjected to serial blind passages in VDS cells
The rCDV-eGFP cDNA clone was co-transfected with three helper plasmids for rescuing the rCDV-eGFP

Summary

Introduction

Canine distemper is a severe infectious disease, affecting a broad variety of domestic and wild carnivores (McCarthy et al, 2007). Canine distemper virions are enveloped and pleomorphic particles containing singlestranded RNA with negative polarity. A CDV genome contains six transcriptional units, independently encoding six structural proteins (N, P, M, F, H and L proteins). The V protein is expressed via an RNA editing strategy from a P gene transcription unit (Mahapatra et al, 2003). All genes are arranged in an order of 3’-N-P/V/C-M-F-H-L-5’ in the CDV genome. The L protein is an RNA-dependent RNA polymerase (RdRp), which is the largest of the virus proteins and is the least abundant. It is assumed to carry all activities necessary for genomic RNA transcription and replication, as well as to be able to cap, methylate and polyadenylate viral mRNAs (Barrett et al, 2006). The morbilliviral L protein can only perform its function as the RdRp, when it associates with its co-factor, the P protein

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