Isolation and Analysis of Rare Norovirus Recombinants from Coinfected Mice Using Drop-Based Microfluidics

Abstract

Human noroviruses (HuNoVs) are positive-sense RNA viruses that can cause severe, highly infectious gastroenteritis. HuNoV outbreaks are frequently associated with recombination between circulating strains. Strain genotyping and phylogenetic analyses show that noroviruses often recombine in a highly conserved region near the junction of the viral polyprotein (open reading frame 1 [ORF1]) and capsid (ORF2) genes and occasionally within the RNA-dependent RNA polymerase (RdRP) gene. Although genotyping methods are useful for tracking changes in circulating viral populations, they report only the dominant recombinant strains and do not elucidate the frequency or range of recombination events. Furthermore, the relatively low frequency of recombination in RNA viruses has limited studies to cell culture or in vitro systems, which do not reflect the complexities and selective pressures present in an infected organism. Using two murine norovirus (MNV) strains to model coinfection, we developed a microfluidic platform to amplify, detect, and recover individual recombinants following in vitro and in vivo coinfection. One-step reverse transcriptase PCR (RT-PCR) was performed in picoliter drops with primers that identified the wild-type and recombinant progenies and scanned for recombination breakpoints at ϳ1-kb intervals. We detected recombination between MNV strains at multiple loci spanning the viral protease, RdRP, and capsid ORFs and isolated individual recombinant RNA genomes that were present at a frequency of 1/300,000 or higher. This study is the first to examine norovirus recombination following coinfection of an animal and suggests that the exchange of RNA among viral genomes in an infected host occurs in multiple locations and is an important driver of genetic diversity. IMPORTANCERNA viruses increase diversity and escape host immune barriers by genomic recombination. Studies using a number of viral systems indicate that recombination occurs via template switching by the virus-encoded RNA-dependent RNA polymerase (RdRP). However, factors that govern the frequency and positions of recombination in an infected organism remain largely unknown. This work leverages advances in the applied physics of drop-based microfluidics to isolate and sequence rare recombinants arising from the coinfection of mice with two distinct strains of murine norovirus. This study is the first to detect and analyze norovirus recombination in an animal model. R ecombination contributes to viral diversity and emergence through the exchange of large portions of genetic material (1). Viruses with segmented RNA genomes, such as influenza virus, are shuffled and reassorted to create new virus subspecies (2). Viruses with linear RNA genomes also recombine, but the mechanism is less clear. Current evidence supports a copy choice mechanism in which viral polymerases, including RNA-dependent RNA polymerase (RdRP) and reverse transcriptase (RT), move between coinfecting viral RNAs (1, 3, 4). Several factors have limited our knowledge of the mol...

Updated classification of norovirus genogroups and genotypes

“…Table 1. Continued Continued recombination breakpoints have been identified across the norovirus genome, they are most frequently found in the ORF1-ORF2 junction region [29][30][31][32][33][34][35][36]. Dual typing (ORF1-RdRp=P type, ORF2=genotype) is now used routinely in many laboratories worldwide and at least 14 GI P-types and 27 GII P-types as well as 9 GI capsid genotypes and 22 GII capsid genotypes have been described [2,19,27].…”

A Cell-based Fluorescence Resonance Energy Transfer (FRET) Sensor Reveals Inter- and Intragenogroup Variations in Norovirus Protease Activity and Polyprotein Cleavage

“…From the data presented here, the GI and GII polyproteins appeared to be cleaved similarly, with both proteases showing similar activity on the heterologous substrates, suggesting that recombination within the polyprotein would not necessarily be restricted by differences in polyprotein cleavage. Recent work by Zhang et al ( 44 ) on two MNV strains has demonstrated the generation in vivo of a variety of recombinants throughout the polyprotein region. Notably, the two strains used (CW3 and WU20) possessed highly similar cleavage sites.…”

Artifact‐Free Quantification and Sequencing of Rare Recombinant Viruses by Using Drop‐Based Microfluidics

“…We modified the above assay to determine the concentration of recombinants resulting from a macrophage cell‐culture co‐infected with a mixture of both parental viruses, each with a multiplicity of infection of 2. We use a one‐step differential RT‐PCR cocktail where each of the two primers is specific to only one parental virus, in order to selectively amplify those recombinants where template switching occurs in a selected 1205 bp region (Figure A; the top row of the gels confirms that our cocktail amplified recombinants from the co‐infection without amplifying parental viruses in the viral mixture). For our drop‐based assay, the differential RT‐PCR cocktail was co‐encapsulated with viral templates, and in‐drop RT‐PCR was performed off‐chip.…”

“…Interestingly, Drop‐9 also includes a synonymous transversion at nucleotide 5221. Using this method, the first recombinant (from Drop‐1 sequence) was also detected from in vivo samples by examining the feces of mice that were co‐infected with both parental strains …”