Generation of porcine reproductive and respiratory syndrome virus by in vitro assembly of viral genomic cDNA fragments

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent for a swine disease affecting the pig industry worldwide. Infection with PRRSV leads to reproductive complications, respiratory illness, and weak immunity to secondary infections. To better control PRRSV infection, novel approaches for generating control measures are critically needed. Here, in vitro Gibson assembly (GA) of viral genomic cDNA fragments was tested for its use as a quick and simple method to recover infectious PRRSV in cell culture. GA involves the activities of T5-exonuclease, Phusion polymerase, and Taq ligase to join overlapping cDNA fragments in an isothermal condition. Four overlapping cDNA fragments covering the entire PRRSV genome and one vector fragment were used to create a plasmid capable of expressing the PRRSV genome. The assembled product was used to transfect a co-culture of 293T and MARC-145 cells. Supernatants from the transfected cells were then passaged onto MARC-145 cells to rescue infectious virus particles. Verification and characterization of the recovered virus confirmed that the GA protocol generated infectious PRRSV that had similar characteristics to the parental virus. This approach was then tested for the generation of a chimeric virus. By replacing one of the four genomic fragments with that of another virus strain, a chimeric virus was successfully recovered via GA. In conclusion, this study describes for the first time the use of GA as a simple, yet powerful tool for generating infectious PRRSV needed for studying PRRSV biology and developing novel vaccines.

Home-made enzymatic premix and Illumina sequencing allow for one-step Gibson assembly and verification of virus infectious clones

“…Based on several reports of infectious clone assembly and reverse genetics b The sequencing depth (read number, orange) and error rate (gray) of the TVMV genome assembled are plotted. c Dot plots show significant DNA alignments between pLX-TVMV (this study, GenBank: MW027845) and pLX-B2 (GenBank: KY825137) or a TVMV reference genome (GenBank: X04083); axis ticks indicate 1-kb intervals of viruses (Pasin et al 2014;Bordat et al 2015;Suhardiman et al 2015), we describe the procedures required for home-made preparation of cloning materials for GA. We successfully applied the home-made enzymatic premix and bacterial competent cells in one-step assembly of a T-DNA binary vector that includes the infectious cDNA clone of an RNA virus. Our cloning strategy was streamlined by omission of slow-growing cloning chassis (e.g.…”

Deciphering the biology of porcine epidemic diarrhea virus in the era of reverse genetics

“…This is accomplished with three key components: (a) a 5 exonuclease to generate recessed ends, (b) DNA polymerase to fill in the gaps after annealing of sticky ends, and (c) DNA ligase. While Gibson assembly has not been applied to coronavirus infectious clone construction, it has been used to successfully recover infectious clones of viruses such as porcine reproductive and respiratory syndrome virus (PRSSV) (Suhardiman et al, 2015), dengue virus (Siridechadilok et al, 2013), and West Nile virus (Vandergaast et al, 2014). Given that it eliminates the need for restriction site design, it may prove to be a very effective tool to complement in vitro ligation as well as cloning into BAC or vaccinia vectors.…”

“…As a ligation method, Gibson assembly does not inherently overcome the issue of sequence instability and toxicity. However, beyond being simply a tool for rapid assembly of cDNA, it can also circumvent the need for bacterial transformation as demonstrated by Suhardiman et al (Suhardiman et al, 2015) and Siridechadilok et al (Siridechadilok et al, 2013), where Gibson assembly reactions were directly transfected into cells for virus production. By generating cDNA by RT-PCR from field samples, this approach can be extremely useful for rapid generation of viral clones that preserve viral sequence diversity.…”

Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning

“…They allow high-throughput simultaneous and scarless assembly of multiple DNA fragments into a plasmid vector in a single reaction using 3′- to 5′-exonuclease to generate complementary single-stranded DNA overhangs in the insert and vector sequences in vitro . Because they are flexible, time-saving, and highly efficient, these sequence- and ligation-independent methods have been used successfully to reconstruct several RNA virus genomes, including influenza A virus [ 45 ], dengue virus [ 46 ], West Nile virus [ 47 ], porcine reproductive and respiratory syndrome virus [ 48 ], LMV [ 12 ], and tomato blistering mosaic virus (ToBMV) [ 49 ]. However, they have not been used in the construction of infectious cDNA clones for PLDMV so far.…”