title: 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 ∼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 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.
  
  
  OBJECTIVE
  RNA 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.
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, China.
authors.forename: Huidan
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
authors.forename: Shelley K
authors.initials: SK
authors.lastname: Cockrell
authors.affiliation: Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Abimbola O
authors.initials: AO
authors.lastname: Kolawole
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Assaf
authors.initials: A
authors.lastname: Rotem
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Adrian W R
authors.initials: AW
authors.lastname: Serohijos
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA Chemical and Biological Engineering Department, Montana State University, Bozeman, Montana, USA.
authors.forename: Connie B
authors.initials: CB
authors.lastname: Chang
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA School of Mechatronics Engineering, Harbin Institute of Technology, Harbin, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Tao
authors.affiliation: Applied Physics Laboratory, Johns Hopkins University, Laurel, Maryland, USA.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Mehoke
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Yulong
authors.initials: Y
authors.lastname: Han
authors.affiliation: Applied Physics Laboratory, Johns Hopkins University, Laurel, Maryland, USA.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Lin
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
authors.forename: Nicholas S
authors.initials: NS
authors.lastname: Giacobbi
authors.affiliation: Applied Physics Laboratory, Johns Hopkins University, Laurel, Maryland, USA.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Feldman
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Eugene
authors.initials: E
authors.lastname: Shakhnovich
authors.affiliation: School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA Department of Physics, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Weitz
authors.affiliation: Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Christiane E
authors.initials: CE
authors.lastname: Wobus
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA pipas@pitt.edu.
authors.forename: James M
authors.initials: JM
authors.lastname: Pipas
mesh_terms: D000818:Animals; D017250:Caliciviridae Infections / Q000821:virology*; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D051379:Mice; D044085:Microfluidics; D008969:Molecular Sequence Data; D029322:Norovirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D010802:Phylogeny; D011995:Recombination, Genetic