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.

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+point+mutation+in+the+rhesus+rotavirus+VP4+protein+generated+through+a+rotavirus+reverse+genetics+system+attenuates+biliary+atresia+in+the+murine+model.">A point mutation in the rhesus rotavirus VP4 protein generated through a rotavirus reverse genetics system attenuates biliary atresia in the murine model.</a> Journal of Virology. 91 (15), 00510-00517 (2017).</li><li>Navarro, A., Trask, S. D., Patton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+genetically+stable+recombinant+rotaviruses+containing+novel+genome+rearrangements+and+heterologous+sequences+by+reverse+genetics.">Generation of genetically stable recombinant rotaviruses containing novel genome rearrangements and heterologous sequences by reverse genetics.</a> Journal of Virology. 87, 6211-6220 (2013).</li><li>Duarte, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotavirus+infection+alters+splicing+of+the+stress-related+transcription+factor+XBP1.">Rotavirus infection alters splicing of the stress-related transcription factor XBP1.</a> Journal of Virology. 93 (5), 01739-01818 (2019).</li><li>Philip, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+recombinant+rotavirus+expressing+NSP3-UnaG+fusion+protein+by+a+simplified+reverse+genetics+system.">Generation of recombinant rotavirus expressing NSP3-UnaG fusion protein by a simplified reverse genetics system.</a> Journal of Virology. , 1616-1619 (2019).</li><li>Papa, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+rotaviruses+rescued+by+reverse+genetics+reveal+the+role+of+NSP5+hyperphosphorylation+in+the+assembly+of+viral+factories.">Recombinant rotaviruses rescued by reverse genetics reveal the role of NSP5 hyperphosphorylation in the assembly of viral factories.</a> Journal of Virology. , (2019).</li><li>Komoto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+genetics+system+demonstrates+that+rotavirus+nonstructural+protein+NSP6+is+not+essential+for+viral+replication+in+cell+culture.">Reverse genetics system demonstrates that rotavirus nonstructural protein NSP6 is not essential for viral replication in cell culture.</a> Journal of Virology. 91 (21), 00695-00717 (2017).</li><li>Philip, A. 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The authors have nothing to disclose.

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 point mutations.
The conditions and incubation times given in this protocol apply to the recovery of well growing strains of recombinant viruses. Adjustments should be considered if attempting to recover SA11 viruses that, due to genetic modification, may be expected to grow poorly. In particular, because the titer of such viruses in transfected BHK-T7/MA014 cell lysates may be low, we typically double the amount of lysate used as inoculum in subsequent amplification steps. Moreover, at the amplification step, poorly growing viruses may require longer times of incubation before reaching levels of CPE sufficient for cell harvesting. Indeed, with such viruses, we may allow infections to proceed for 10&#8722;14 days, or even longer, before harvesting cells. Finally, poorly growing viruses are likely to generate small, slow-growing plaques on MA104 cells. Thus, to plaque isolate these viruses, it may be necessary to allow plaques to develop until 6&#8722;10 days post infection before staining cells with neutral red and picking plaques.
In our experience, the single most important factor in the reliable recovery of the recombinant rotavirus is the use of healthy, well-maintained BHK-T7 cells. In our laboratory, we routinely passage BHK-T7 cells 2x a week at the same dilution using medium supplemented not only with 10% FBS, but also with NEAA, TPB, and high levels of glucose (GMEM complete medium). The additional supplements help BHK-T7 cells retain extended viability following plasmid transfection, a factor likely crucial for recovering poorly growing mutant recombinant viruses. We supplement the medium every other passage with G418, an antibiotic which selects for maintenance of the T7 polymerase expression plasmid. Passage conditions should be such that BHK-T7 cells are never allowed to grow past confluency, conditions that rapidly lead to decreased cell viability. For us, BHK-T7 cells that have been allowed to overgrow perform poorly in the reverse genetics protocol, even if the cells are subsequently passaged appropriately multiple times. Instead of attempting to rehabilitate overgrown BHK-T7 cells, we restart the lineage with cells previously stored in liquid nitrogen.
Mycoplasma contamination of BHK-T7 and MA104 cells can be a major factor in the failure of the rotavirus reverse genetics system to generate recombinant virus. In our laboratory, we use a PCR-based mycoplasma detection kit (Table of Materials) to check for contamination of cell lines, and when detected, it is most often associated with our BHK-T7 cells. We have not attempted to cure cell lines of contaminating mycoplasma, instead re-establishing the lines with earlier mycoplasma-free passages stored in liquid nitrogen. Prior to starting new cell lines, we discard all previously used medium and medium supplements and thoroughly decontaminate incubators, biological safety cabinets, water baths, lab benches, and pipettors. We also use mycoplasma detection kits to check stocks of recombinant viruses for contamination. Because of the resistance of the rotavirus particles to denaturation by organic solvents, such as Vertrel VF32, it is possible to free virus stocks from contaminating mycoplasma, negating the need to regenerate recombinant viruses by reverse genetics. It is important to stress that cell lines and virus preparations received into the laboratory should be checked for mycoplasma contamination prior to routine use.
Although day-in and day-out, we use the same protocol to generate recombinant viruses, we know that certain modifications can be made which will not preclude virus recovery. For example, (i) co-transfection of the pCMV/NSP868R capping-enzyme plasmid with SA11 pT7 vectors is not required for recovery of recombinant viruses. While addition of the capping plasmid yields higher virus titers in transfected BHK-T7/MA104 cell lysates, we have been able to recover numerous viruses, including those expressing FPs, without it. However, we have concluded that expression of the capping enzyme by pCMV/NP868R may contribute significantly to the recovery of less fit viruses. (ii) We have found that recombinant viruses can be generated even if the amount of the transfection reagent (Table of Materials) used in the reverse genetics protocol is reduced by one-half, a modification that can significantly reduce expenses. (iii) Similarly, we have determined that M199 complete medium can be used in place of DMEM complete medium. (iv) Finally, there is no set requirement concerning the type of vector backbone that must be used in producing SA11 T7 transcription vectors. As long as the viral cDNA in the plasmid is surrounded by an upstream T7 promoter and a downstream HDV ribozyme and T7 terminator, the plasmid can be expected to support the recovery of recombinant virus. Notably, pGEM-, pBluescript, and pUC-based vectors have been used successfully in the reverse genetics system.

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&#8722;D, F&#8722;J) have been defined3. Those strains comprising rotavirus species A are responsible for the vast majority of human disease4. The introduction of rotavirus vaccines into childhood immunization programs beginning in the last decade is correlated with significant reductions in rotavirus mortality and morbidity. Most notably, the number of rotavirus-associated childhood deaths has decreased from approximately 528,000 in 2000 to 128,500 in 20164,5. Rotavirus vaccines are formulated from live attenuated strains of the virus, with 2 to 3 doses administered to children by 6 months of age. The large number of genetically diverse rotavirus strains circulating in humans and other mammalians species, combined with their ability to rapidly evolve through mutagenesis and reassortment, may lead to antigenic changes in the types of rotaviruses infecting children6,7,8. Such changes may undermine the efficacy of existing vaccines, requiring their replacement or modification.
The development of fully plasmid-based reverse genetics systems enabling manipulation of any of the 11 rotavirus genome segments was only recently achieved9. With the availability of these systems, it has become possible to unravel molecular details of rotavirus replication and pathogenesis, to develop improved high-throughput screening methods for anti-rotavirus compounds, and to create new potentially more effective classes of rotavirus vaccines. During rotavirus replication, capped viral (+)RNAs not only guide the synthesis of viral proteins, but also serve as templates for the synthesis of progeny dsRNA genome segments10,11. All rotavirus reverse genetics systems described to date rely on the transfection of T7 transcription vectors into mammalian cell lines as a source of cDNA-derived (+)RNAs used in recovering recombinant viruses9,12,13. Within the transcription vectors, full-length viral cDNAs are positioned between an upstream T7 promoter and downstream hepatitis delta virus (HDV) ribozyme such that viral (+)RNAs are synthesized by T7 RNA polymerase that contain authentic 5&#8217; and 3&#8217;-termini (Figure 1A). In the first-generation reverse genetics system, recombinant viruses were made by transfecting baby hamster kidney cells expressing T7 RNA polymerase (BHK-T7) with 11 T7 (pT7) transcription vectors, each directing synthesis of a unique (+)RNA of the simian SA11 virus strain, and three CMV promoter-drive expression plasmids, one encoding the avian reovirus p10FAST fusion protein and two encoding subunits of the vaccinia virus D1R-D12L capping enzyme complex9. Recombinant SA11 viruses generated in transfected BHK-T7 cells were amplified by overseeding with MA104 cells, a cell line permissive for rotavirus growth. A modified version of the first-generation reverse genetics system has been described that no longer uses support plasmids12. Instead, the modified system successfully generates recombinant rotaviruses simply by transfecting BHK-T7 cells with the 11 SA11 T7 transcription vectors, with the caveat that vectors for the viral factory (viroplasm) building blocks (nonstructural proteins NSP2 and NSP5) are added at levels 3-fold higher than the other vectors14,15. Modified versions of the reverse genetics system have also been developed that support the recovery of the human KU and Odelia strains of rotavirus16,17. The rotavirus genome is remarkably amenable to manipulation by reverse genetics, with recombinant viruses generated to date with mutations introduced into VP418, NSP19, NSP219, NSP320,21, and NSP522,23. Among the most useful viruses generated so far are those that have been engineered to express fluorescent reporter proteins (FPs)9,12,21,24,25.
In this publication, we provide the protocol for the reverse genetics system that we use in our laboratory to generate recombinant strains of SA11 rotavirus. The key feature of our protocol is co-transfection of BHK-T7 cells with the 11 pT7 transcription vectors (modified to include 3x levels of the pT7/NSP2SA11 and pT7/NSP5SA11 vectors) and a CMV expression vector encoding the African swine fever virus (ASFV) NP868R capping enzyme21 (Figure 2). In our hands, presence of the NP868R plasmid leads to the production of higher titers of recombinant viruses by transfected BHK-T7 cells. In this publication, we also provide a protocol for modifying the pT7/NSP3SA11 plasmid such that recombinant viruses can be generated that express not only the segment 7 protein product NSP3 but also a separate FP. This is accomplished by re-engineering the NSP3 open reading frame (ORF) in the pT7/NSP3SA11 plasmid to contain a downstream 2A translational stop-restart element followed by an FP ORF (Figure 1B)24,26. Through this approach, we have generated recombinant rotaviruses expressing various FPs: UnaG (green), mKate (far-red), mRuby (red), TagBFP (blue), CFP (cyan), and YFP (yellow)24,27,28. These FP-expressing rotaviruses are made without deleting the NSP3 ORF, thus yielding viruses that are expected to encode a full complement of functioning viral proteins.

<table><tbody><tr><td>Baby Hamster Kidney - T7 RdRP (BHK-T7) Cells</td><td></td><td></td><td>Contact: ubuchholz@niaid.nih.gov</td></tr><tr><td>Bio-Rad 8-16% Tris-Glycine Polyacrylamide Mini-Gel</td><td>Bio-Rad</td><td>45608105</td><td></td></tr><tr><td>Cellometer AutoT4 viable cell counter</td><td>Nexcelom</td><td></td><td></td></tr><tr><td>ChemiDoc MP Gel Imaging System</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Chloroform</td><td>MP</td><td>194002</td><td></td></tr><tr><td>Clarity Western Enhanced Chemiluminescence (ECL) Substrate</td><td>Bio-Rad</td><td>170-5060</td><td></td></tr><tr><td>Competent E.coli DH5alpha Bacteria</td><td>Lucigen</td><td>60602-2</td><td></td></tr><tr><td>Complete Protease Inhibitor</td><td>Pierce</td><td>A32965</td><td></td></tr><tr><td>Disposable Transfer Pipettes, Ultrafine Extended Tips</td><td>MTC Bio</td><td>P4113-11</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Lonza</td><td>12-604F</td><td></td></tr><tr><td>Eagle&#039;s Minimal Essential Medium, 2x (2xEMEM)</td><td>Quality Biological</td><td>115-073-101</td><td></td></tr><tr><td>Ethanol, Absolute (200 proof)</td><td>Fisher Bioreagents</td><td>BP2818-500</td><td></td></tr><tr><td>Ethidium Bromide Solution (10 mg/ml)</td><td>Invitrogen</td><td>15585-011</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Corning</td><td>35-010-CV</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), Heat Inactivated</td><td>Corning</td><td>35-011-CV</td><td></td></tr><tr><td>Flag M2 Antibody, Mouse Monoclonal</td><td>Sigma-Aldrich</td><td>F1804</td><td></td></tr><tr><td>GenEluate HP Plasmid Midiprep Kit</td><td>Sigma</td><td>NA0200-1KT</td><td></td></tr><tr><td>Geneticin (G-418)</td><td>Invitrogen</td><td>10131-027</td><td></td></tr><tr><td>Gibco FluroBrite DMEM</td><td>ThermoFisher</td><td>A1896701</td><td>DMEM with low background fluorescence</td></tr><tr><td>Glasgow Minimal Essential Medium (GMEM)</td><td>Gibco</td><td>11710-035</td><td></td></tr><tr><td>Goat Anti-Rabbit IgG, Horseradish Peroxidase (HRP) Conjugated</td><td>Cell-Signaling Technology</td><td>7074S</td><td></td></tr><tr><td>Guinea Pig Anti-NSP3 Antiserum</td><td>Patton lab</td><td>lot 55068</td><td></td></tr><tr><td>Guinea Pig Anti-VP6 Antierum</td><td>Patton lab</td><td>lot 53963</td><td></td></tr><tr><td>Horse Anti-Guinea Pig IgG, Horseradish Peroxidase (HRP) Conjugated</td><td>KPL</td><td>5220-0366</td><td></td></tr><tr><td>Horse Anti-Mouse IgG, Horseradish eroxidase (HRP) Conjugated</td><td>Cell-Signaling Technology</td><td>7076S</td><td></td></tr><tr><td>iNtRON Biotechnology e-Myco Mycoplasma PCR Detection Kit</td><td>JH Science</td><td>25235</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>Macron</td><td>3032-02</td><td></td></tr><tr><td>L-glutamine Solution (100x)</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>Luria Agar Powder (Miller&#039;s LB Agar)</td><td>RPI research products</td><td>L24020-2000.0</td><td></td></tr><tr><td>Medium 199 (M199) Culture Medium</td><td>Hyclone</td><td>Sh30253.01</td><td></td></tr><tr><td>Minimal Essential Medium -Eagle Joklik&#039;s Forumation (SMEM)</td><td>Lonza</td><td>04-719Q</td><td></td></tr><tr><td>Monkey Kidney (MA104) Cells</td><td>ATCC</td><td>ATCC CRL-2378.1</td><td></td></tr><tr><td>NanoDrop One Spectrophotometer</td><td>ThermoScientific</td><td></td><td></td></tr><tr><td>Neutral Red Solution (0.33%)</td><td>Sigma-Aldrich</td><td>N2889-100ml</td><td></td></tr><tr><td>Non-Essential Amino Acid Solution (100x)</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>Novex 10% Tris-Glycine Polyacrylamide Mini-Gel</td><td>Invitrogen</td><td>XP00102BOX</td><td></td></tr><tr><td>Nuclease-Free Molecular Biology Grade Water</td><td>Invitrogen</td><td>10977-015</td><td></td></tr><tr><td>NucleoSpin Gel and PCR Clean-Up Kit</td><td>Takara</td><td>740609.25</td><td></td></tr><tr><td>Opti-MEM Reduced Serum Medium</td><td>Gibco</td><td>31985-070</td><td></td></tr><tr><td>Pellet pestle (RNase-free, disposable)</td><td>Fisher</td><td>12-141-368</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution, (100x penn-strep)</td><td>Corning</td><td>30-002-Cl</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS), 10x</td><td>Fisher Bioreagents</td><td>BP399-20</td><td></td></tr><tr><td>Porcine Trypsin, Type IX-S</td><td>Sigma-Aldrich</td><td>T0303</td><td></td></tr><tr><td>PureYield Plasmid Miniprep System</td><td>Promega</td><td>A1223</td><td></td></tr><tr><td>Qiagen Plasmid Maxi Kit</td><td>Qiagen</td><td>12162</td><td></td></tr><tr><td>Qiagen Plasmid Midi Kit</td><td>Qiagen</td><td>12143</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>SA11 pT7 Transcription Vectors</td><td>Addgene</td><td>89162-89172</td><td></td></tr><tr><td>SA11 pT7/NSP3 Transcription Vectors Expressing Fluorescent Proteins</td><td></td><td></td><td>Contact: jtpatton@iu.edu</td></tr><tr><td>SeaKem LE Agarose</td><td>Lonza</td><td>50000</td><td>For gel electrophoresis</td></tr><tr><td>SeaPlaque agarose</td><td>Lonza</td><td>50100</td><td>For plaque assay</td></tr><tr><td>Superscript III One-Step RT-PCR kit</td><td>Invitrogen</td><td>12574-035</td><td></td></tr><tr><td>Trans-Blot Turbo Nitrocellulose Transfer Kit</td><td>Bio-Rad</td><td>170-4270</td><td></td></tr><tr><td>Trans-Llot Turbo Transfer System</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>TransIT-LTI Transfection Reagent</td><td>Mirus</td><td>MIR2306</td><td></td></tr><tr><td>Tris-Glycine-SDS Gel Running Buffer (10x)</td><td>Bio-Rad</td><td>161-0772</td><td></td></tr><tr><td>Triton X 100</td><td>Fisher Bioreagents</td><td>BP151-500</td><td></td></tr><tr><td>Trizol RNA Extraction Reagent</td><td>Ambion</td><td>15596026</td><td></td></tr><tr><td>Trypan blue</td><td>Corning</td><td>25-900-CI</td><td></td></tr><tr><td>Trypsin (0.05%)-EDTA (0.1%) Cell Dissociation Solution</td><td>Quality Biological</td><td>118-087-721</td><td></td></tr><tr><td>Tryptose Phosphate Broth</td><td>Gibco</td><td>18050-039</td><td></td></tr><tr><td>Tween-20</td><td>VWR</td><td>0777-1L</td><td></td></tr><tr><td>Vertrel VF solvent</td><td>Zoro</td><td>G0707178</td><td></td></tr><tr><td>Zoe Fluorescent Live Cell Imager</td><td>Bio-Rad</td><td></td><td></td></tr></tbody></table>

simplified reverse genetics method to recover recombinant rotaviruses expressing reporter proteins

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.

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, yielding titers of recombinant wildtype SA11 virus (rSA11/wt) in BHK-T7/MA104 cell lysates of ~104 PFU/mL and in amplified MA104 cell lysates of &#62;1 x 107 PFU/mL. SA11 recombinant viruses generated by reverse genetics using modified pT7/NSP3SA11 plasmids expressing FPs (e.g., rSA11/NSP3-2A-3xFL-UnaG) grow to titers that are ~4-fold less than rSA11/wt.
Following the reverse genetics protocol, we generated recombinant SA11 viruses that were easily identified by plaque assay on MA104 cells, thus allowing plaque isolation (Figure 3D). Viruses in plaques were picked with a long-tip disposable transfer pipet and amplified on MA104 cells. The dsRNA genomes of plaque-purified rSA11/wt and rSA11/NSP3-2A-3xFL-UnaG viruses were extracted with guanidinium thiocyanate, resolved by electrophoresis on a 10% polyacrylamide gel, and detected by staining with ethidium bromide (Figure 3A). As expected, the segment 7 (NSP3) dsRNA of rSA11/NSP3-2A-3xFL-UnaG migrated much slower than that of rSA11/wt (Figure 3A) due to the presence of 2A-3xFL-UnaG sequences. The segment 7 (NSP3) dsRNA of rSA11/NSP3-2A-3xFL-UnaG was gel purified, converted to cDNA form by RT-PCR, and sequenced to confirm accuracy.
To check for expression of the UnaG fluorescent protein, MA104 cells in 6-well plates were infected with 3 PFU per cell of rSA11/wt and rSA11/NSP3-2A-3xFL-UnaG. At 8 h post infection, culture medium in plates were replaced with 0.5 mL of DMEM with low background fluorescence per well and the plates incubated for an additional 30 min at 37 &#176;C in a CO2 incubator. Afterwards, plates were examined for UnaG expression using a live cell imager. The analysis showed that rSA11/NSP3-2A-3xFL-UnaG produced green fluorescence, verifying the functionality of the UnaG gene in the recombinant virus (Figure 3B). In contrast, green fluorescence was not detected in cells infected with rSA11/wt. To address whether the 2A element in the modified segment 7 of rSA11/NSP3-2AxFL-UnaG promoted the expression of two separate proteins (NSP3-2A and 3xFL-UnaG), MA104 cells were infected with rSA11/NSP3-2A-3xFL-UnaG and rSA11/wt. Cell lysates were prepared from cells harvested at 8 h post infection, resolved by gel electrophoresis, and blotted onto nitrocellulose filters. The blots were probed with antibodies specific for rotavirus VP6 and NSP3, and FLAG tag. The analysis showed that NSP3-2A and 3xFL-UnaG were expressed as separate proteins in cells infected with rSA11/NSP3-2A-3xFL-UnaG, indicating that the 2A element was functional (Figure 3C). Cells infected with rSA11/wt did not express protein recognized by anti-FLAG antibody. The NSP3 protein present in rSA11/NSP3-2A-3xFL-UnaG-infected cells by anti-NSP3 antibody migrated slightly slower than NSP3 present in rSA11/wt-infected cells due to the presence of remnant 2A residues at the C-terminus of NSP3.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61039/61039fig1.jpg" /> 
Figure 1: Rotavirus reverse genetics plasmids. (A) Full-length cDNAs of the 11 SA11 rotavirus genome segments are positioned within pT7 plasmids, ligated upstream with a promoter for T7 RNA polymerase and downstream with an HDV ribozyme. In the presence of T7 RNA polymerase, the rotavirus pT7 plasmids produce full-length SA11 (+)RNAs with authentic 5&#8217; and 3&#8217; termini. (B) Schematics of the (+)RNA and protein products made by the pT7/NSP3-2A-3xFL-UnaG plasmid. The schematic includes the NSP3 cDNA sequence, and sequences for the porcine teschovirus 2A-like (2A) element, the 3x FLAG (FL) tag, and the green fluorescent protein UnaG. The position of the 2A translational stop-restart site is indicated with a red arrow. Due to the activity of the 2A element, translation of the RNA produces two proteins. The NSP3 portion contains remnants of the 2A element and the UnaG portion is fused to a 3x FLAG tag. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61039/61039fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61039/61039fig2.jpg" /> 
Figure 2: Rotavirus reverse genetics system. BHK-T7 monolayers are transfected with 11 pT7 plasmids, each expressing a different SA11 (+)RNA, and a pCMV vector expressing the African swine fever virus (ASFV) NP868R capping enzyme (pCMV/NP868R). At 3 days post infection (d.p.i.), the BHK-T7 cells are overseeded with MA104 cells. At 7 days post infection, recombinant rotaviruses in BHK-T7/MA104 cell lysates are amplified by passage on MA104 cells, then isolated by plaque purification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61039/61039fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61039/61039fig3.jpg" /> 
Figure 3: Characteristics of the recombinant strains rSA11/wt and rSA11/NSP3-2A-3xFL-UnaG. (A) Electrophoretic profiles of the dsRNA genome segments of plaque-isolated rSA11 strains. The 11 genome segments are numbered and the shift in the position of segment 7 (NSP3) is indicated with a red line. (B) Fluorescence detected in rSA11-infected MA104 cells at 8 h post infection using a live cell imager (20x magnification) set on the green detection channel. Scale bar = 100 &#181;m. (C) Immunoblot analysis of proteins present at 8 h post infection in MA104 cells infected with rSA11 strains using guinea pig anti-VP6 and anti-NSP3 antisera and mouse anti-FLAG monoclonal antibody. (D) Plaques produced by rSA11/wt on MA104 cells at 3 days post infection and detected by neutral red staining. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61039/61039fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins at JoVE.com

Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins

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.

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian ...

simplified-reverse-genetics-method-to-recover-recombinant-rotaviruses

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.5K Views.  Indiana University. Dit protocol beschrijft een betrouwbaar en efficiënt omgekeerd geneticasysteem voor het maken van recombinant rotavirussen. Het legt ook uit hoe recombinant rotavirussen die fluorescerende marker eiwitten uit te drukken. Deze methode is eenvoudig die een minimaal aantal plasmiden en kan genereren recombinant rotavirussen die afzonderlijke fluor eiwitten uitdrukken, alsmede ook virale eiwitten.Begin met het zaaien van BHKT7 cellen op 12-well platen. Spoel een vers samenvloeiende monola...

Video: Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus in the family Bunyaviridae and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3 as well as wild-type RVFV strains 4-6, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8 and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10 IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.

The reverse genetics system for the Rift Valley fever virus MP-12 vaccine strain is a useful tool for creating additional MP-12 mutants with increased attenuation and immunogenicity. We describe the protocol to generate and characterize NSs mutant strains.

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal ...

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA viruses. By integrating a full RNA genome error-prone PCR and reverse genetics, random genome-wide substitution mutagenesis can be induced. We have developed a method using this technique to synthesize diverse libraries to identify viral mutants with phenotypes of interest. This method, called full-length mutant RNA synthesis (FL-MRS), offers the following advantages: (i) the ability to create a large library via a highly efficient one-step error-prone PCR; (ii) the ability to create groups of libraries with varying levels of genetic diversity by manipulating the fidelity of DNA polymerase; (iii) the creation of a full-length PCR product that can directly serve as a template for mutant RNA synthesis; and (iv) the ability to create RNA that can be delivered into host cells as a non-selected input pool to screen for viral mutants of the desired phenotype. We have found, using a reverse genetics approach, that FL-MRS is a reliable tool to study viral-directed evolution at all stages in the life cycle of the hepatitis C virus, JFH1 isolate. This technique appears to be an invaluable tool to employ directed evolution to understand adaptation, replication, and the role of viral genes in pathogenesis and antiviral resistance in positive-sense RNA viruses.

The protocol describes a method for introducing controllable genetic diversity in the hepatitis C virus genome by combining full-length mutant RNA synthesis using error-prone PCR and reverse genetics. The method provides a model for phenotype selection and can be used for 10 kb long positive-sense RNA virus genomes.

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA ...

Genetics

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV&#8217;s easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host range selection method coupled with visual identification of recombinant viruses. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. The cassette, including the gene of interest and the mCherry-E3L fusion is flanked by sequences derived from the VACV genome. Between the gene of interest and mCherry-E3L is a smaller region that is identical to the first ~150 nucleotides of the 3&#8217; arm, to promote homologous recombination and loss of the mCherry-E3L gene after selection. We demonstrate that this method permits efficient, seamless generation of rVACV in a variety of cell types without requiring drug selection or extensive screening for mutant viruses.

This is a method to generate &#8220;scarless&#8221; recombinant vaccinia viruses using host-range selection and visual identification of recombinant viruses.

Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins

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