Analysis of Synchronous and Asynchronous In Vitro Infections with Homologous Murine Norovirus Strains Reveals Time-Dependent Viral Interference Effects.

Louisa F Ludwig-Begall,Chiara Ceci,Elisabetta Di Felice,Axel Mauroy,Barbara Toffoli,Fulvio Marsilio,Etienne Thiry,Barbara Di Martino

doi:10.3390/v13050823

Abstract

Viral recombination is a key mechanism in the evolution and diversity of noroviruses. In vivo, synchronous single-cell coinfection by multiple viruses, the ultimate prerequisite to viral recombination, is likely to be a rare event and delayed secondary infections are a more probable occurrence. Here, we determine the effect of a temporal separation of in vitro infections with the two homologous murine norovirus strains MNV-1 WU20 and CW1 on the composition of nascent viral populations. WU20 and CW1 were either synchronously inoculated onto murine macrophage cell monolayers (coinfection) or asynchronously applied (superinfection with varying titres of CW1 at half-hour to 24-h delays). Then, 24 h after initial co-or superinfection, quantification of genomic copy numbers and discriminative screening of plaque picked infectious progeny viruses demonstrated a time-dependent predominance of primary infecting WU20 in the majority of viral progenies. Our results indicate that a time interval from one to two hours onwards between two consecutive norovirus infections allows for the establishment of a barrier that reduces or prevents superinfection.

Highlights

Human noroviruses (HuNoVs) are recognised as a leading global cause of sporadic and epidemic viral gastroenteritis [1] and account for a global economic burden of $60 billion, over one million hospitalisations, and 200,000 deaths per annum [2,3]
The linear, polyadenlyated 7.4–7.7 kb long HuNoV genome is organised into three open reading frames (ORFs); an additional fourth ORF is described for murine norovirus (MuNoV) [20,21]
To quantitatively assess viral progeny distributions 24 h after initial co-or superinfection, MNV-1 WU20 and CW1 genomic copy numbers were inferred from the cycle threshold (Ct) values of the quantitative PCR (qPCR) reactions and normalised against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Ct values

Summary

Introduction

Human noroviruses (HuNoVs) are recognised as a leading global cause of sporadic and epidemic viral gastroenteritis [1] and account for a global economic burden of $60 billion, over one million hospitalisations, and 200,000 deaths per annum [2,3]. Various HuNoV infection models have yielded valuable insights into the NoV life cycle in recent years [9,10,11,12]. Many of these experimental systems are technically challenging and as yet lack the degree of robustness required for detailed decipherment. The 5 proximal NoV ORF1 encodes a large polyprotein that is co-and post-translationally cleaved into six non-structural viral proteins [22]. ORF2 and ORF3 encode the structural virion components, major and minor capsid proteins, VP1 and VP2, respectively. ORF4, which entirely overlaps the 5 end of ORF2, encodes virulence factor (VF1) [23]

Methods

Results

Conclusion