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

Abstract

Porcine epidemic diarrhoea virus (PEDV) causes acute diarrhoea and dehydration in swine of all ages, with significant mortality in neonatal pigs. The recent rise of PEDV outbreaks in Asia and North America warrants an urgent search for effective vaccines. However, PEDV vaccine research has been hampered by difficulties in isolating and propagating the virus in mammalian cells, thereby complicating the recovery of infectious PEDV using a full-length infectious clone. Here, we engineered VeroE6 cells to stably express porcine aminopeptidase N (pAPN) and used them as a platform to obtain a high-growth variant of PEDV, termed PEDV AVCT12 . Subsequently, the full-length cDNA clone was constructed by assembling contiguous cDNA fragments encompassing the complete genome of PEDV AVCT12 in a bacterial artificial chromosome. Infectious PEDV could be recovered, and the rescued virus displayed phenotypic properties identical to the parental virus. Interestingly, we found that PEDV AVCT12 contained a C-terminal deletion of the spike gene, resulting in disruption of the ORF3 start codon. When a functional ORF3 gene was restored, the recombinant virus could not be rescued, suggesting that ORF3 could suppress PEDV replication in vitro. In addition, a high-growth and genetically stable recombinant PEDV expressing a foreign protein could be rescued by replacing the ORF3 gene with the mCherry gene. Together, the results of this study provide a means to generate genetically defined PEDV as a promising vaccine candidate.

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

“…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). In 2016, Beall et al constructed infectious cDNA clones of a highly pathogenic US PEDV strain PC22A using in vitro ligation of contiguous cDNA fragments, and performed in vitro transcription to generate infectious viral RNA (Beall et al, 2016).…”

Porcine gastroenteric viruses: pathogenesis and protection

“…This isolate most likely grows in vivo in gnotobiotic piglets, as animals seroconverted after inoculation (Lin et al, 2015b), but its virulence remains to be determined.It has been shown that during the generation of live attenuated vaccines for PEDV by passing the virus in Vero cells, in the case of both the Korean DR13strain and the Japanese P-5V attenuated viruses included a protein 3 in which more than half of the carboxyterminal domain was deleted . Similarly, while a recombinant virus lacking ORF3 expression could be rescued, when ORF3 was restored the recombinant virus could not be rescued (Jengarn et al, 2015). Therefore, it seems that protein 3 could have a role in vivo and that its total or partial deletion helps to maintain PEDV in Vero cells.…”

“…Âî âðåìÿ âñïûøêè âèðóñíîé èíôåêöèè ïðåîáëàäàþùèì íàïðàâëåíèåì âåòðà â òå÷åíèå ìåñÿöà áûëî ñåâåðíîå. Èíîãäà âåòðû äóëè â þãî-çàïàäíîì (Jengarn et al, 2015). Although PEDV is in general propagated in Vero cells, it efficiently infects a variety of cell types, including porcine intestinal epithelial cells (IECs) (Zhao et al, 2014)or human Huh 7 cells (Liu et al, 2015).…”

“…Two PEDV infectious cDNA clones have been reported, providing the necessary tools for the engineeringof genetically attenuated PEDVs as vaccine candidates (Beall et al, 2016;Jengarn et al, 2015). Jengarn and collaborators have engineered a full-length cDNA clone that was constructed by assembling contiguous cDNA fragments encompassing the complete PEDV genome in a bacterial artificial chromosome (BAC), following techniques previously described by our laboratory (Almazan et al, 2000;Almazan et al, 2015).…”

Virulence factors in porcine coronaviruses and vaccine design

“…The first CoV infectious cDNA clone was obtained for TGEV, as a bacterial artificial chromosome (BAC) (Almazan et al, 2000). Since then, additional cDNA clones were obtained for TGEV and PEDV, based either on BACs or in vitro ligation (Almazan et al, 2014; Beall et al, 2016; Jengarn et al, 2015). This review will focus on the study of enteric CoVs interaction with the host to determine virulence genes, since the modification or deletion of these genes may lead to virus attenuation, and to the generation of vaccine candidates to prevent infection by porcine CoVs.…”