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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Generation of recombinant rotaviruses from plasmid DNA provides an essential tool for the study of rotavirus replication and pathogenesis, and the development of rotavirus expression vectors and vaccines. Herein, we describe a simplified reverse genetics approach for generating recombinant rotaviruses, including strains expressing fluorescent reporter proteins.

Abstract

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian and avian host species, including humans. With the recent advent of rotavirus reverse genetics systems, it has become possible to use directed mutagenesis to explore rotavirus biology, modify and optimize existing rotavirus vaccines, and develop rotavirus multitarget vaccine vectors. In this report, we describe a simplified reverse genetics system that allows the efficient and reliable recovery of recombinant rotaviruses. The system is based on co-transfection of T7 transcription vectors expressing full-length rotavirus (+)RNAs and a CMV vector encoding an RNA capping enzyme into BHK cells constitutively producing T7 RNA polymerase (BHK-T7). Recombinant rotaviruses are amplified by overseeding the transfected BHK-T7 cells with MA104 cells, a monkey kidney cell line that is highly permissive for virus growth. In this report, we also describe an approach for generating recombinant rotaviruses that express a separate fluorescent reporter protein through the introduction of a 2A translational stop-restart element into genome segment 7 (NSP3). This approach avoids deleting or modifying any of the viral open reading frames, thus allowing the production of recombinant rotaviruses that retain fully functional viral proteins while expressing a fluorescent protein.

Introduction

Rotaviruses are major causes of severe gastroenteritis in infants and young children, as well as the young of many other mammalian and avian species1. As members of the Reoviridae family, rotaviruses have a segmented double-stranded RNA (dsRNA) genome. The genome segments are contained within a nonenveloped icosahedral virion formed from three concentric layers of protein2. Based on sequencing and phylogenetic analysis of the genome segments, nine species of rotavirus (A−D, F−J) have been defined3. Those strains comprising rotavirus species A are responsible for the vast major....

Protocol

1. Media preparation and cell culture maintenance

  1. Obtain baby hamster kidney cells constitutively expressing T7 RNA polymerase (BHK-T7) and African green monkey kidney MA104 cells.
    NOTE: BHK-T7 (or BSR-T7) cells are not commercially available, but are a common cell line of laboratories using reverse genetics to study RNA virus biology. The BHK-T7 cell line used in this protocol was obtained from Dr. Ursula J. Buchholz (National Institutes of Health, Bethesda, MD, USA), a co-developer of the original BH.......

Representative Results

The reverse genetics protocol described in this article proceeds through multiple distinct steps: (1) co-transfection of BHK-T7 cells with rotavirus pT7 transcription vectors and a pCMV/NP868R expression plasmid, (2) overseeding of transfected BHK-T7 cells with MA104 cells, (3) amplification of recombinant viruses present in BHK-T7/MA104 cells lysates using MA104 cells, and (4) plaque isolation of recombinant virus using MA104 cells (Figure 2). In our hands, the protocol is efficient, yieldi.......

Discussion

In our laboratory, we routinely rely on the reverse genetics protocol described herein to produce recombinant SA11 rotaviruses. With this approach, individuals with little experience in molecular biology techniques or working with rotaviruses recover recombinant viruses even on their first attempt. We have generated close to 100 recombinant viruses following this protocol, including those with genomes that have been re-engineered to express foreign proteins (e.g., FPs) and that contain sequence additions, deletions, and .......

Acknowledgements

This work was supported by NIH grants R03 AI131072 and R21 AI144881, Indiana University Start-Up Funding, and the Lawrence M. Blatt Endowment. We thank members of the IU Rotahoosier laboratory, Ulrich Desselberger, and Guido Papa for their many contributions and suggestions in developing the reverse genetics protocol.

....

Materials

NameCompanyCatalog NumberComments
Baby Hamster Kidney - T7 RdRP (BHK-T7) CellsContact: ubuchholz@niaid.nih.gov
Bio-Rad 8-16% Tris-Glycine Polyacrylamide Mini-GelBio-Rad45608105
Cellometer AutoT4 viable cell counterNexcelom
ChemiDoc MP Gel Imaging SystemBio-Rad
ChloroformMP194002
Clarity Western Enhanced Chemiluminescence (ECL) SubstrateBio-Rad170-5060
Competent E.coli DH5alpha BacteriaLucigen60602-2
Complete Protease InhibitorPierceA32965
Disposable Transfer Pipettes, Ultrafine Extended TipsMTC BioP4113-11
Dulbecco's Modified Eagle Medium (DMEM)Lonza12-604F
Eagle's Minimal Essential Medium, 2x (2xEMEM)Quality Biological115-073-101
Ethanol, Absolute (200 proof)Fisher BioreagentsBP2818-500
Ethidium Bromide Solution (10 mg/ml)Invitrogen15585-011
Fetal Bovine Serum (FBS)Corning35-010-CV
Fetal Bovine Serum (FBS), Heat InactivatedCorning35-011-CV
Flag M2 Antibody, Mouse MonoclonalSigma-AldrichF1804
GenEluate HP Plasmid Midiprep KitSigmaNA0200-1KT
Geneticin (G-418)Invitrogen10131-027
Gibco FluroBrite DMEMThermoFisherA1896701DMEM with low background fluorescence
Glasgow Minimal Essential Medium (GMEM)Gibco11710-035
Goat Anti-Rabbit IgG, Horseradish Peroxidase (HRP) ConjugatedCell-Signaling Technology7074S
Guinea Pig Anti-NSP3 AntiserumPatton lablot 55068
Guinea Pig Anti-VP6 AntierumPatton lablot 53963
Horse Anti-Guinea Pig IgG, Horseradish Peroxidase (HRP) ConjugatedKPL5220-0366
Horse Anti-Mouse IgG, Horseradish eroxidase (HRP) ConjugatedCell-Signaling Technology7076S
iNtRON Biotechnology e-Myco Mycoplasma PCR Detection KitJH Science25235
Isopropyl alcoholMacron3032-02
L-glutamine Solution (100x)Gibco25030-081
Luria Agar Powder (Miller's LB Agar)RPI research productsL24020-2000.0
Medium 199 (M199) Culture MediumHycloneSh30253.01
Minimal Essential Medium -Eagle Joklik's Forumation (SMEM)Lonza04-719Q
Monkey Kidney (MA104) CellsATCCATCC CRL-2378.1
NanoDrop One SpectrophotometerThermoScientific
Neutral Red Solution (0.33%)Sigma-AldrichN2889-100ml
Non-Essential Amino Acid Solution (100x)Gibco11140-050
Novex 10% Tris-Glycine Polyacrylamide Mini-GelInvitrogenXP00102BOX
Nuclease-Free Molecular Biology Grade WaterInvitrogen10977-015
NucleoSpin Gel and PCR Clean-Up KitTakara740609.25
Opti-MEM Reduced Serum MediumGibco31985-070
Pellet pestle (RNase-free, disposable)Fisher12-141-368
Penicillin-Streptomycin Solution, (100x penn-strep)Corning30-002-Cl
Phosphate Buffered Saline (PBS), 10xFisher BioreagentsBP399-20
Porcine Trypsin, Type IX-SSigma-AldrichT0303
PureYield Plasmid Miniprep SystemPromegaA1223
Qiagen Plasmid Maxi KitQiagen12162
Qiagen Plasmid Midi KitQiagen12143
QIAprep Spin Miniprep KitQiagen27104
SA11 pT7 Transcription VectorsAddgene89162-89172
SA11 pT7/NSP3 Transcription Vectors Expressing Fluorescent ProteinsContact: jtpatton@iu.edu
SeaKem LE AgaroseLonza50000For gel electrophoresis
SeaPlaque agaroseLonza50100For plaque assay
Superscript III One-Step RT-PCR kitInvitrogen12574-035
Trans-Blot Turbo Nitrocellulose Transfer KitBio-Rad170-4270
Trans-Llot Turbo Transfer SystemBio-Rad
TransIT-LTI Transfection ReagentMirusMIR2306
Tris-Glycine-SDS Gel Running Buffer (10x)Bio-Rad161-0772
Triton X 100Fisher BioreagentsBP151-500
Trizol RNA Extraction ReagentAmbion15596026
Trypan blueCorning25-900-CI
Trypsin (0.05%)-EDTA (0.1%) Cell Dissociation SolutionQuality Biological118-087-721
Tryptose Phosphate BrothGibco18050-039
Tween-20VWR0777-1L
Vertrel VF solventZoroG0707178
Zoe Fluorescent Live Cell ImagerBio-Rad

References

  1. Crawford, S. E., et al. Rotavirus infection. Nature Reviews Disease Primers. 3 (17083), 1-16 (2017).
  2. Settembre, E. C., Chen, J. Z., Dormitzer, P. R., Grigorieff, N., Harrison, S. C. Atomic model of an infectious rotavirus particle. The EMBO Journal<....

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