Construction, Characterization and Application of Recombinant Porcine Deltacoronavirus Expressing Nanoluciferase.

Puxian Fang,Jie Ren,Sijin Xia,Shaobo Xiao,Gang Wang,Liurong Fang,Jiansong Zhang,Huichang Zhang,Liyuan Tian,He Sun

doi:10.3390/v13101991

Abstract

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV.

Highlights

Coronavirus (CoVs) infections usually causes respiratory and gastroenteritis illness in humans or animals, and are widely known for their capacity to transmit across species, such as severe acute respiratory syndrome (SARS-CoV), Middle East respiratory syndrome (MERS-CoV) from the human pathogens, and the recently-emerged SARS-CoV-2, and the animal pathogens porcine deltacoronavirus (PDCoV) and swine enteric alphacoronavirus (SeACoV) [1,2,3]
In accordance with the Immunofluorescence Assay (IFA) results, Western blot assay demonstrated that NS6 expression was not observed in Nluc reporter virus-infected cells, and N protein expression was observed in all PDCoVs-infected cells (Figure 4D). These results demonstrate the successful recovery of Nluc reporter virus, rCHN-HN-2014-4NS6-Nluc
We further evaluated the effect of lycorine and resveratrol on the wild type PDCoV CHN-HN2014 strain and demonstrated that the infection of CHN-HN-2014 strain was significantly inhibited by lycorine treatment, but not by resveratrol treatment, in line with these results of their effect on the infection of Nluc reporter virus (Figure 6G,F).These results showed that Nluc reporter virus is suitable for high throughput screening antivirals due to its quantifiable feature

Summary

Introduction

Coronavirus (CoVs) infections usually causes respiratory and gastroenteritis illness in humans or animals, and are widely known for their capacity to transmit across species, such as severe acute respiratory syndrome (SARS-CoV), Middle East respiratory syndrome (MERS-CoV) from the human pathogens, and the recently-emerged SARS-CoV-2, and the animal pathogens porcine deltacoronavirus (PDCoV) and swine enteric alphacoronavirus (SeACoV) [1,2,3]. These pathogenic coronaviruses have posed a great threat to public health and animal health. PDCoV was subsequently reported in many states [13] and other countries, such as South Korea [14,15], mainland China [16,17,18], Thailand, Laos, Vietnam [19,20,21], and

Methods

Results

Conclusion