Isolation and analysis of rare norovirus recombinants from co-infected XXXX using drop-based microfluidics.

@entity1 @entity19 @entity1902 @entity325 @entity65851 @entity34503
UNASSIGNED: @entity1 noroviruses (HuNoVs) are positive-sense RNA viruses that can cause severe, highly infectious @entity1902 . 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 ( @entity34503 ) and capsid ( @entity65851 ) genes and occasionally within the RNA-dependent RNA polymerase (RdRP) gene. Although genotyping methods are useful for tracking changes in circulating viral populations, they only report 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 that do not reflect the complexities and selective pressures present in an infected organism. Using two @entity19 norovirus (MNV) strains to model @entity325 , we developed a microfluidic platform to amplify, detect, and recover individual recombinants following in vitro and in vivo @entity325 . One-step RT-PCR was performed in picoliter drops with primers that identified the wild-type and recombinant progenies and scanned for recombination breakpoints at approximately 1-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 greater. This study is the first to examine norovirus recombination following @entity325 of an animal and suggests that the exchange of RNA among viral genomes in the infected host occurs in multiple locations and is an important driver of genetic diversity. IMPORTANCE: RNA viruses increase diversity and escape host immune barriers by genomic recombination. Studies from 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 @entity325 of @entity19 with two distinct strains of @entity19 norovirus. This report is the first to detect and analyze norovirus recombination in an animal model.
