Porcine epidemic diarrhea virus (PEDV), a swine enteric coronavirus causing acute diarrhea in piglets, is one of the major threatens to the pork industry globally. Reverse genetics is a valuable tool for the virological study and vaccine development for coronaviruses. Due to the large size and unstable problem in Escherichia coli of coronavirus genome, construction and manipulation of reverse genetics system for coronaviruses remain laborious and time-consuming. In this study, a reverse genetics system of the genotype II PEDV strain HM was generated using the transformation-associated recombination (TAR) technology in yeast within 1 week. The rescued virus (rPEDV) exhibited similar growth properties to the wild-type virus in vitro. With this PEDV infectious cDNA clone, CRISPR/Cas9 technology and homologous recombination were combined to generate a recombinant virus rPEDV-EGFP in which the ORF3 gene was swapped with an EGFP gene. The reporter virus displayed similar growth properties to the parental virus rPEDV and remained stable during serial passage in vitro. Of note, the strategies of construction and manipulation of PEDV infectious cDNA clone are extremely simple and efficient, which could be applied for other RNA viruses and DNA viruses.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that poses a substantial threat to the global swine industry. However, our current understanding of the host factors crucial for PEDV infection remains limited. To identify these host factors, we conducted a genome-wide CRISPR/Cas9 gene knockout screen using a PEDV-permissive cell line. Our results indicate that the endogenous expression of human interferon-inducible transmembrane protein 3 (IFITM3) enhances PEDV entry and replication. Silencing or eliminating endogenous IFITM3 in Huh7 cells significantly suppressed PEDV entry, whereas reintroducing IFITM3 partially restored susceptibility to PEDV. Overexpression of human IFITM3 or IFITM2, but not IFITM1, in Huh7.5 cells substantially increased PEDV entry and replication. Importantly, our results suggest that human IFITM3 influences PEDV entry at a later stage. Furthermore, the overexpression of porcine IFITM1 significantly enhanced PEDV infection in LLC-PK1 cells, whereas the overexpression of porcine IFITM2/3 did not produce similar effects. Notably, removing the C-terminal 15 amino acids of porcine IFITM2/3 resulted in increased PEDV entry. Coimmunoprecipitation analyses showed that all IFITMs interacted with the PEDV S1 protein, indicating a direct role in the viral entry process. Additionally, porcine IFITM1 colocalized with the PEDV S protein at the cell nuclear periphery and enhanced PEDV infection in porcine small intestinal organoids. Overall, our results suggest that IFITMs are critical in facilitating PEDV entry into cells. Targeting IFITMs may provide a promising strategy for controlling PEDV transmission and developing interventions to mitigate the virus’s impact on the swine industry. IMPORTANCE: Understanding the mechanisms underlying porcine epidemic diarrhea virus (PEDV) infection is vital for addressing its significant impact on the swine industry. This study reveals that interferon-inducible transmembrane (IFITM) proteins, particularly human IFITM3 and porcine IFITM1, play crucial roles in facilitating PEDV entry and replication. By elucidating these molecular interactions, the research highlights the potential of IFITMs as therapeutic targets for managing PEDV infections and paves the way for antiviral strategies. Moreover, this research extends beyond PEDV management, underscoring the critical role of host factors in controlling the spread of pathogenic coronaviruses.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens to the swine industry worldwide over the past three decades. No approved effective antiviral drug is available to control this virus. The antiviral effects of allicin (diallyl thiosulfinate) on many human and animal viruses have been documented. However, the antiviral effect of allicin on PRRSV infection remains unknown. In this study, we found that allicin exhibited an inhibitory effect on HP-PRRSV and NADC30-like PRRSV in a dose-dependent manner by interfering with viral entry, replication, and assembly. Furthermore, allicin alleviated the expression of pro-inflammatory cytokines (IFN-β, IL-6, and TNFα) induced by PRRSV infection. The pro-inflammatory signaling pathways, TNF signaling pathway and MAPK signaling pathway, up-regulated by PRRSV infection were restored by allicin treatment. Taken together, these results demonstrate that allicin has antiviral activity against PRRSV and ameliorates inflammatory responses induced by PRRSV infection, suggesting that allicin is a promising drug candidate for anti-PRRSV therapy in vivo.

Porcine epidemic diarrhea virus (PEDV), a member of the genera alphacoronavirus, causes acute watery diarrhea and dehydration in suckling piglets and results in enormous economic losses in the swine industry worldwide. Identification and characterization of different cell lines are not only invaluable for PEDV entry and replication studies but also important for the development of various types of biological pharmaceuticals against PEDV. In this study, we present an approach to identify suitable permissive cell lines for PEDV research. Human cell lines were screened for a high correlation coefficient with the established PEDV infection model Huh7 based on RNA-seq data from the Cancer Cell Line Encyclopedia (CCLE). Experimentally testing permissiveness towards PEDV infection, three highly permissive human cell lines, HepG2, Hep3B217, and SNU387 were identified. The replication kinetics of PEDV in HepG2, Hep3B217, and SNU387 cells were similar to that in Vero and Huh7 cells. Additionally, the transcriptomes analysis showed robust induction of transcripts associated with the innate immune in response to PEDV infection in all three cell lines, including hundreds of inflammatory cytokine and interferon genes. Moreover, the expression of inflammatory cytokines and interferons were confirmed by qPCR assay. Our findings indicate that HepG2, Hep3B217, and SNU387 are suitable cell lines for PEDV replication and innate immune response studies.

The reverse genetics system is a valuable tool in the virological study of RNA viruses. With the availability of reverse genetics, the porcine reproductive and respiratory syndrome virus (PRRSV) has been utilized as a viral vector for the expression of foreign genes of interest. Here, we constructed a full-length cDNA clone of a highly pathogenic PRRSV (HP-PRRSV) TA-12 strain. Using this cDNA clone, we generated a reporter virus expressing a gaussia luciferase (Gluc) via an additional subgenomic RNA between ORF7 and 3′UTR. This reporter virus exhibited similar growth kinetics to the wild-type (WT) virus and remained genetically stable for at least ten passages in MARC-145 cells. In cells infected with this reporter virus, the correlation between the expression levels of Gluc in culture media and the virus titers suggested that Gluc is a good indicator of the reporter virus infection. With this reporter virus, we further established the Gluc readout-based assays for antiviral drug screening and serum neutralizing antibody detection that exhibited comparable performance to the classical assays. Taken together, we established a reverse genetics system of HP-PRRSV and generated a novel reporter virus that could serve as a valuable tool for antiviral drug screening and serum neutralizing antibody detection.