Devils Eye (Mirus (1.2))
To wash, add 0. Place QIAquick column into a clean 1. Ligate the corresponding fragments with related backbones. Transform into the Top 10 competent cells. Verify the inserts by DNA sequencing. Assemble the whole genome following Fig. Vortex for 5 s and centrifuge for 1 min at 13, rpm. Note: After centrifuge, there are two layers in the liquid and gently take out the tube from centrifuge.
Take off the top layer carefully and transfer to a new tube. Back extraction: Add pL ddH 2 0 into the previous tube including phenol:chloroform; vortex for 5 s and centrifuge for 1 min at 13, rpm.
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Therefore, the total of liquid volume is pL. Add equal volume of chloroform pL , vortex for 5 s, and centrifuge at 13, rpm for 10 min. Take off the top layer and transfer to another new tube. Load 1.
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Thaw the frozen reagents. Mix thoroughly. The white pellet should be seen. Warm up PBS, trypsin, and medium. Prechill a 4 mm cuvette and a bottle of PBS, and keep them on ice. Thaw 10 pg of viral RNA on ice. Remove supernatant from monolayer Vero cells and wash with warm PBS once. After centrifugation, remove supernatant, and disperse cells by tapping the tube with finger gently see Note 7.
Add 10 pg RNA to the cuvette. Transfer 8 x 10 6 cells pL to a prechilled 4 mm cuvette, and keep cuvette on ice. Mix gently. Perform electroporation using the Gene Pulser 0. Leave the electroporated cells at room temperature for 10 min. Seed the cells into the 8-well chambers to check the viral protein expression.
Follow Subheading 3. Do not remove media from more than one well plate at once in case the cells were dried. To prevent the overlay detaching the cells, do not add it directly on middle of the well. Instead add the overlay on the side of the wall. If the color of the mixture is orange or violet, add 10 pL of 3 M sodium acetate, pH 5. The color of the mix- 58 Chao Shan et al. Elution efficiency is dependent on pH.
The maximum elution efficiency is achieved between pH 7. The spermidine in the lOx reaction buffer can coprecipitate the template DNA if the reaction is assembled on ice. Add the lOx reaction buffer after the water and the ribonucleotides are already in the tube. Do not let the pellet dry as it is hard to dissolve the RNA when it is dried. Do not use pipette to disperse the cells as they are fragile now. Howley ed , Fields virology, 6th edn, vol 1. Emerg Infect Dis 21 2 Euro Surveill 19 14 5. ACS Infect Dis 6.
N Engl J Med. J Virol 76 12 Antivir Res 91 1 : J Gen Virol 95 Pt 4 Rapid intervention necessitates the capacity to generate, grow, and genetically manipulate infectious CoVs in order to rapidly evaluate pathogenic mechanisms, host and tissue permissibility, and candidate antiviral therapeutic efficacy. CoVs encode the largest viral RNA genomes at about , nucleotides in length, and thereby complicate efficient engineering of the genome.
Deconstructing the genome into manageable fragments affords the plasticity necessary to rapidly introduce targeted genetic changes in parallel and assort mutated fragments while maximizing genome stability over time.
In this protocol we describe a well-developed reverse genetic platform strategy for CoVs that is comprised of partitioning the viral genome into independent DNA fragments depending on the CoV genome , each subcloned into a plasmid for increased stability and ease of genetic manipulation and amplification.
Coronavirus genomes are conveniently partitioned by introducing type IIS or IIG restriction enzyme recognition sites that confer directional cloning. Since each restriction site leaves a unique overhang between adjoining fragments, reconstruction of the full-length genome can be achieved through a standard DNA ligation comprised of equal molar ratios of each fragment.
Using this method, recombinant CoVs can be rapidly generated and used to investigate host range, gene function, pathogenesis, and candidate therapeutics for emerging and preemergent CoVs both in vitro and in vivo. Cockrell et al. The portent of a worldwide pandemic mobilized the scientific community, leading to robust public health intervention strategies that controlled the epidemic. Because of the availability of reverse genetics, robust in vitro replication, and in vivo animal models of human disease, SARS-CoV has become the most intensively studied prototype for HCoV research .
MERS-CoV-infected individuals have also traveled internationally, illustrating the potential for global spread. Camels are suspected to be intermediate hosts between bats and humans that can repeatedly allow for reemergence of MERS-CoV in the human population. Blue indicates the publication of RGS clones.
Importantly, much of the HCoV research over the last 15 years has been possible because of the capacity to generate infectious clones using highly efficient reverse genetics platforms , coupled with robust small animal models of human disease [17, 18]. Reverse genetic systems for coronaviruses were difficult to achieve because of the large size of the viral RNA genome, genome stability in bacterial vectors, difficulty in driving full-length 30 kb RNA transcripts in vitro, poor transfection efficiencies, and low infectivity of the viral genome.
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In , the first molecular clone was developed for transmissible gastroenteritis virus TGEV , using targeted splice junctions to increase genome stability in low- copy baculovirus vectors  Fig. A few months later, our group published an alternative TGEV reverse genetic strategy , and then again applied this technique for the group 2 murine coronavirus [21 ]. A final innovation in CoV molecular clone design was the insertion of full-length HCoV E molecular clone into vaccinia virus in . After in vitro transcription and transfection of full-length genomes into permissive cells, recombinant viruses are recovered which contain the genetic content of the molecular clone.
Although the reverse genetic system RGS described here achieved prominence shortly after the emergence of SARS-CoV , this platform has been used to generate CoV infectious clones that span nearly the entire breadth of the Coronaviridae family, including pathogenic viruses from groups la and lb of the alphacoromiviruses and groups 2a, 2b, and 2c of the betacoronavi- ruses Fig.
For type IIS e. Thus, these enzymes leave unique ends, providing directionality during multi-segment assembly. Moreover, the recognition site is not palindromic, allowing for seamless assembly of component cDNA clones into full-length genes and genomes.
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Q to to td. Bacterial Amplification 2. Plasmid Digestion 3. Fragment Ligation 5. Viral RtJA Transcription 6. A second approach uses type IIG e. In this instance, the restriction site is retained in the assembled product. Fragments are then resolved on an agarose gel, purified, and ligated Fig.
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In some instances, strong T7 stop sites are mutated to promote full-length transcript synthesis in in vitro transcription reactions . Resulting genome- length mRNA is electroporated into a permissive mammalian cell line Fig. Cloning success and viral fitness can be measured by plaque assay and growth curves Fig. Northern blot image is reprinted from .
Both of these methods can be used to confirm productive CoV replication following RGS clone generation Finally, our group has applied this approach to generate stable molecular clones for flaviviruses that include dengue and the newly emerged Zika virus [31, 32]. Revere Genetics of Emerging Coronaviruses 67 1. For example, an early application included the introduction of over 27 mutations into the SARS-CoV genome at 9 different genome transcription regulatory sequences, thereby demonstrating for the first time that the transcription regulatory circuit of a virus could be rewired .
In a seminal pathogenesis study RGS was used to validate that six mutations acquired during mouse adaptation of the SARS-CoV Urbani MAI 5 strain indeed caused lung pathology associated with severe acute respiratory distress syndrome [ 18 ]. Since these mutations were dispersed across the entire genome, RGS proved to be an efficient method to introduce all six mutations simultaneously in order to generate a robust mouse-adapted SARS-CoV strain.
After nearly a decade of research the MAI5 strain continues to play a dominant role in SARS-CoV mouse pathogenesis studies, including vaccine and therapeutic evaluations [7, 17, ]. Notably, SARS-CoVgain-of-function GOF studies have yielded invaluable information regarding the role of viral proteins in pathogenesis in animal models and in tissue culture studies [17, 36]. Applying the combined technologies of GOF studies with the RGS will be essential to future research on emergent and preemergent coronaviruses.
The following protocol outlines the RGS used to generate infectious clones of MERS-CoV, and provides detailed methods for building recombinant coronaviruses using this technique. Chemically competent bacterial cells Top 10, Invitrogen. Qiaprep Spin Miniprep Kit Qiagen. Gel electrophoresis-grade agarose. Ethidium bromide, or other DNA-visualizing dye.
DNA ladder that allows for determination of fragment size. DNA gel electrophoresis equipment.