This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.
Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.
Zika virus (ZIKV; Family Flaviviridae: Genus Flavivirus) is a mosquito-borne flavivirus that arrived in Brazil in 2013-14 and was subsequently associated with a massive outbreak of febrile illness that spread throughout the Americas1. In addition, ZIKV has been linked with severe disease outcomes, such as Guillain-Barré syndrome in adults and microcephaly in fetuses and neonates2. Little was known about ZIKV before its rapid spread in the western hemisphere. This included a lack of molecular tools, thus hindering mechanistic research. Molecular tools for viruses, such as infectious cDNA clones, facilitate vaccine and antiviral therapeutic development, and allow for the assessment of viral genetic factors related to differential viral pathogenesis, immune response and viral evolution.
Flavivirus infectious clones are known to be highly unstable in bacteria due to cryptic prokaryotic promoters present in their genomes3. Several approaches have been used to ameliorate this problem; including insertion of tandem repeats upstream of viral sequences4, mutation of putative prokaryotic promoter sequences5, splitting the genome into multiple plasmids6, low-copy number vectors (including bacterial artificial chromosomes)7,8 and insertion of introns in the viral genome9. One full-length system without modifications has been described for ZIKV; however, this clone appeared to be attenuated in cell culture and in mice10. Other groups have engineered introns into the ZIKV genome, allowing for disruption of unstable sequences in bacteria that can be spliced out in mammalian cells in vitro for the production of infectious virus11,12. Additionally, a PCR-based system entitled Infectious-Subgenomic-Amplicons has been used successfully to rescue the prototype MR766 strain of ZIKV13. The approach described here requires no foreign sequences, but rather disrupts the genome at the region of high instability by using multiple plasmids, which has previously been used successfully with yellow fever6, dengue14,15 and West Nile viruses16. Furthermore, the addition of the hepatitis D ribozyme sequence at the viral genomic termini facilitates the creation of an authentic 3' end without the need for the addition of a linearization site. Additionally, both plasmids are constructed in a low-copy number vector (pACYC177, ~15 copies per cell) to mitigate any residual toxicity17. The virus recovered displays a growth profile comparable to parental virus in in vitro growth curves in 8 cell lines comprising a variety of cell-types derived from both mammals and insects, and has exhibited identical pathogenic profiles in mice and infection, dissemination and transmission rates in mosquitoes18.
Herein, we detail a protocol describing how to grow the infectious clone plasmids, generate full-length viral RNA (the viral genome) in vitro, and recover infectious virus in cell culture. First, we describe the propagation of plasmids in bacteria or bacteria-free amplification using rolling circle amplification (RCA). Next, we show how the two plasmids are digested and then subsequently ligated together to generate full-length virus. Finally, we describe the production of transcribed RNA and its subsequent electroporation into Vero cells, followed by titration of recovered virus (Figure 1). The approach described is rapid, allowing for recovery of infectious virus stocks in 1-2 weeks.
Here we describe a method for the recovery of a bipartite infectious cDNA clone system for ZIKV. Previously described clones for ZIKV suffer from either attenuation or require the addition of introns, making plasmids larger and preventing rescue in insect cells. Infectious virus can be recovered using the two-plasmid clone system in either mammalian or insect cells (data not shown). In addition, virus recovered from this system behaves similarly to wild-type virus in several cell lines, in an immunocompromised mouse mode…
The authors have nothing to disclose.
The authors would like to thank Kristen Bullard-Feibelman, Milena Veselinovic and Claudia Rückert for their assistance in characterizing the clone-derived virus. This work was supported in part by grants from the National Institute of Allergy and Infectious Diseases, NIH under grants AI114675 (BJG) and AI067380 (GDE).
NEB Stable CompetentE. coli | New England BioLabs | C3040H | |
Carbenicillin, Disodium Salt | various | ||
Zyppy Plasmid Miniprep Kit | Zymo Research | D4036 | |
ZymoPURE Plasmid Maxiprep Kit | Zymo Research | D4202 | |
SalI-HF | New England BioLabs | R3138S | 20,000 units/ml |
NheI-HF | New England BioLabs | R3131S | 20,000 units/ml |
ApaLI | New England BioLabs | R0507S | 10,000 units/ml |
EcoRI-HF | New England BioLabs | R3101S | 20,000 units/ml |
BamHI-HF | New England BioLabs | R3136S | 20,000 units/ml |
HindIII-HF | New England BioLabs | R3104S | 20,000 units/ml |
illustra TempliPhi 100 Amplification Kit | GE Healthcare Life Sciences | 25640010 | |
NucleoSpin Gel and PCR Clean-up | Macherey-Nagel | 740609.5 | |
Shrimp Alkaline Phosphatase (rSAP) | New England BioLabs | M0371S | 1,000 units/ml |
Alkaline Phosphatase, Calf Intestinal (CIP) | New England BioLabs | M0290S | 10,000 units/ml |
T4 DNA Ligase | New England BioLabs | M0202S | 400,000units/mL |
HiScribe T7 ARCA mRNA Kit | New England BioLabs | E2065S | |
Vero cells | ATCC | CCL-81 | |
ECM 630 High Throughput Electroporation System | BTX | 45-0423 | Other machines are acceptable. |
LB Broth with agar (Miller) | Sigma | L3147 | Can be homemade as well. |
Terrific Broth | Sigma | T0918 | Can be homemade as well. |
Petri Dish | Celltreat | 229693 | |
Culture Tubes | VWR International | 60818-576 | |
T75 flasks | Celltreat | 229340 | |
T182 flasks | Celltreat | 229350 | |
1x PBS | Corning | 21-040-CV | |
RPMI 1640 with L-glutamine | Corning | 10-040-CV | |
DMEM with L-glutamine and 4.5 g/L glucose | Corning | 10-017-CV | |
Fetal Bovine Serum (FBS) | Atlas Biologicals | FP-0500-A | |
Tragacanth Powder | MP Bio | MP 104792 | |
Crystal Violet | Amresco | 0528-1006 | |
Ethanol Denatured | VWR International | BDH1156-1LP | |
6 well plate | Celltreat | 229106 | |
12 well plate | Celltreat | 229111 | |
Sequencing Oligos | IDT | see table 1 | |
Qubit 3.0 | ThermoFisher | Qubit 3.0 | other methods are acceptable. |
Qubit dsDNA BR Assay Kit | ThermoFisher | Q32850 | other methods are acceptable. |
Qubit RNA HS Assay Kit | ThermoFisher | Q32852 | other methods are acceptable. |
Class II Biosafety Cabinet | Varies | N/A | This is necessary for live-virus work. |