The overall goal of this experimental procedure is the generation of infectious recombinant Zika virus from a full-length cDNA clone, assembled in a bacterial artificial chromosome under the control of the human cytomegalovirus immediate-early promoter. The development of reverse genetic system for RNA viruses such as Zika virus allow the direct manipulation of the virus genome to study multiple aspect of the viral biology and pathogenesis and to develop vaccine strategy. The main advantage of this technique is that the construction of Zika virus infectious clone as a bacterial artificial chromosome overcomes the toxicity associated with the flavivirus genome and allows the rescue of infectious virus by direct transfection of the BAC cDNA clone into susceptible cells.
This methodology could be applied to constructing stable and fully-functional infectious cDNA clones for other flaviviruses as well as other positive-stranded RNA viruses. To begin this procedure, construct the Zika virus cDNA clone as outlined in the text protocol, using appropriated restriction sites. Four DNA fragments spanning the Zika virus genome flanked by the human CMV promoter at the five-prime end and the hepatitis delta virus ribosome and the bovine growth hormone termination and polyadenylation sequences at the three-prime end are assembled in a bacterial artificial chromosome plasmid.
First, grow a single colony of E.coli DH10B carrying the pBAC-Zika virus infectious clone in five milliliters of LB medium containing chloramphenicol for eight hours at 37 degrees Celsius with gentle shaking. Then, add 500 milliliters of the LB medium containing chloramphenicol to a two-liter flask. Add one milliliter of the prepared bacterial culture to the flask and grow the cells at 37 degrees Celsius for 14 to 16 hours until the OD600 is approximately 0.6 to 0.8.
After this, use a commercial kit specifically developed for BAC purification to purify the BAC infection cDNA clone by alkaline lysis by following the manufacturer's instructions. One day before transfection, plate Vero cells into the wells of a six-well plate at a density of 500, 000 cells per well in growth medium. To prepare the transfection mix, add four micrograms of the BAC cDNA clone to 250 microliters of serum reduced medium and mix carefully.
In a separate tube, dilute 12 microliters of transfection agent in 250 microliters of serum reduced medium. Vortex to mix and incubate at room temperature for five minutes. Next, combine the diluted BAC cDNA with the transfection agent.
Mix carefully and incubate at room temperature for 20 to 30 minutes. During this incubation period, use growth medium without antibiotics to wash the Vero cells and leave the cells in one milliliter of fresh medium without antibiotics. Then, distribute the 500 microliters of the cDNA and transfection reagent mixture onto the Vero cells dropwise.
Rock the plate back and forth to mix and incubate the cells in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide for six hours. After this, remove the transfection medium and add two milliliters of fresh growth medium with antibiotics. Incubate the cells in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide, making sure to check every day for induction of cytopathic effect.
After four to six days of transfection, when the CPE is around 50 to 75%collect the tissue culture supernatants in 15-milliliter conical tubes and centrifuge for 10 minutes to remove cellular debris. Then, harvest the supernatants containing recombinant Zika virus and discard the cell pellets. Aliquot the supernatant in cryotubes and store them at minus 80 degrees Celsius.
To begin, grow Vero cells to 90%confluence. Wash the cells two times with PBS and infect them with the rescued virus with the multiplicity of infection of 0.1 in growth medium containing 2%FBS. Place the plates of cells in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide and rock every 15 minutes for the 90-minute adsorption period.
After viral adsorption, remove the viral inoculum and add fresh growth medium with 2%FBS. Incubate three or four days at the same conditions used previously. When the CPE is approximately 75%collect the tissue culture supernatant and centrifuge to remove cellular debris.
Then, harvest the supernatant containing recombinant Zika virus and discard the pellet. Aliquot the supernatant into cryotubes. Place the tubes in a freezing box and store them at minus 80 degrees Celsius.
When ready, remove a virus aliquot from the freezer and determine the viral titer by plaque assay as outlined in the text protocol. In this study, a stable, full-length cDNA clone of the Zika virus strain Rio Grande do Norte Natal is generated by sequentially cloning four overlapping cDNA fragments into the bacterial artificial chromosome pBeloBAC11 using conventional cloning methods and unique restriction sites present in the viral genome. This full-length cDNA clone was flanked at the five-prime end by the human cytomegalovirus promoter to allow the expression of viral RNA in the nucleus of transfected cells and at the three-prime end by the hepatitis delta virus ribosome followed by the bovine growth hormone termination and polyadenylation sequence to produce RNAs containing the exact three-prime end of the viral genome.
Once the BAC cDNA is assembled, infectious virus can be easily recovered after the direct transfection of susceptible Vero cells with the BAC cDNA clone using cationic liposomes. Using this approach, R Zika virus RGN can be rescued with titers higher than one million plaque-forming units per milliliter. In addition, the rescued virus induces a clear cytopathic effect and generates homogenous plaques of about two millimeters in size.
The rescued virus identity is confirmed via immunofluorescence analysis using a specific monoclonal antibody for the Zika virus E protein. Overall, these results demonstrate that infectious recombinant Zika virus can be rescued from full-length cDNA clones assembled in a BAC. In summary, we have developed a powerful Zika virus reverse genetic approach based on the use of a bacterial artificial chromosome that could be adapted to generate the stable and functional infections with cDNA clone of other positive-strand RNA viruses to facilitate the study of the pathology, to develop a vaccine, and or to facilitate the identification of drug with antiviral activity.
