Manipulation of the Porcine Epidemic Diarrhea Virus Genome Using Targeted RNA Recombination

Abstract

Porcine epidemic diarrhea virus (PEDV) causes severe economic losses in the swine industry in China and other Asian countries. Infection usually leads to an acute, often lethal diarrhea in piglets. Despite the impact of the disease, no system is yet available to manipulate the viral genome which has severely hampered research on this virus until today. We have established a reverse genetics system for PEDV based on targeted RNA recombination that allows the modification of the 3′-end of the viral genome, which encodes the structural proteins and the ORF3 protein. Using this system, we deleted the ORF3 gene entirely from the viral genome and showed that the ORF3 protein is not essential for replication of the virus in vitro. In addition, we inserted heterologous genes (i.e. the GFP and Renilla luciferase genes) at two positions in the viral genome, either as an extra expression cassette or as a replacement for the ORF3 gene. We demonstrated the expression of both GFP and Renilla luciferase as well as the application of these viruses by establishing a convenient and rapid virus neutralization assay. The new PEDV reverse genetics system will enable functional studies of the structural proteins and the accessory ORF3 protein and will allow the rational design and development of next generation PEDV vaccines.

Genetic manipulation of porcine epidemic diarrhoea virus recovered from a full-length infectious cDNA clone

“…In contrast to our results, they showed that disruption of ORF3 expression by small interfering RNA resulted in decreased virus production in infected Vero cells. However, the high growth of ORF3-null PEDV shown in this study and by Li et al (2013) disputes the essential role of ORF3 in PEDV replication. It is also worth pointing out that our results so far suggest a strong correlation between ORF3 and cell-culture adaptability.…”

“…However, the recovery of infectious PEDV using the full-length cDNA approach has been challenged by several technical difficulties, one of which is the fact that available cell lines do not adequately support productive replication of the virus, and the full-length cDNA clone of PEDV is not stable in conventionally used plasmid vectors. Notably, Li et al (2013) recently described the reverse genetics of PEDV based on targeted RNA recombination technology. Although applicable, this approach could be complicated by several steps including the generation of a PEDV derivative carrying spikes derived from another coronavirus, and use the RNA of this so-called chimeric virus as an intermediate for recombination with a parental RNA to restore the PEDV spike.…”

“…Our results indicate that not only did this recombinant virus grow with growth kinetics comparable to those of the WT virus, it also exhibited strong expression of mCherry, which could be observed in infected cells even after multiple passages, suggesting that PEDV can tolerate the addition of a foreign gene. Similarly, a study by Li et al (2013) also successfully generated recombinant PEDV expressing foreign proteins including GFP and Renilla luciferease. Interestingly, it has also been shown in this study that, besides the ORF3 reading frame, a foreign gene can be introduced under the transcription regulatory sequences of the S gene.…”

Novel Porcine Epidemic Diarrhea Virus (PEDV) Variants with Large Deletions in the Spike (S) Gene Coexist with PEDV Strains Possessing an Intact S Gene in Domestic Pigs in Japan: A New Disease Situation

“…However, how the sialic acid binding activity affects the tropism of these alphacoronaviruses remains unclear. The S protein N-domain of some PEDV strains were thought to be dispensable for replication of PEDV in vitro [44–46]. The large deletions in the S protein of the PEDV variants in this study indicated that this domain might also be dispensable in vivo .…”

Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology

“…Reverse genetics systems are valuable tools to study the functions of viral genes and to generate recombinant viruses with defined genetic changes as vaccine candidates. In 2013, Li et al first reported a reverse genetics system for the Korean classical PEDV vaccine strain DR13 based on a targeted RNA recombination method (Li et al, 2013). Following this, Jengarn et al engineered an infectious cDNA clone of the Thailand classical PEDV strain AVCT12 into a bacterial artificial chromosome (BAC) using eight contiguous cDNA fragments (Jengarn et al, 2015).…”