Regulatory Non-Coding RNAs during Porcine Viral Infections: Potential Targets for Antiviral Therapy.

Pigs play important roles in agriculture and bio-medicine; however, porcine viral infections have caused huge losses to the pig industry and severely affected the animal welfare and social public safety. During viral infections, many non-coding RNAs are induced or repressed by viruses and regulate viral infection. Many viruses have, therefore, developed a number of mechanisms that use ncRNAs to evade the host immune system. Understanding how ncRNAs regulate host immunity during porcine viral infections is critical for the development of antiviral therapies. In this review, we provide a summary of the classification, production and function of ncRNAs involved in regulating porcine viral infections. Additionally, we outline pathways and modes of action by which ncRNAs regulate viral infections and highlight the therapeutic potential of artificial microRNA. Our hope is that this information will aid in the development of antiviral therapies based on ncRNAs for the pig industry.

Generation of APN-chimeric gene-edited pigs by CRISPR/Cas9-mediated knock-in strategy.

The prevalence of porcine enteric coronaviruses (PECs), including transmissible gastroenteritis virus (TGEV), swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine delta coronavirus (PDCoV), and porcine epidemic diarrhea virus (PEDV), poses a serious threat to animal and public health. Here, we aimed to further optimize the porcine aminopeptidase N (pAPN) gene editing strategy to explore the balance between individual antiviral properties and the biological functions of pAPN in pigs. Finally, APN-chimeric gene-edited pigs were produced through a CRISPR/Cas9-mediated knock-in strategy. Further reproductive tests indicated that these gene-edited pigs exhibited normal pregnancy rates and viability. Notably, in vitro viral challenge assays further demonstrated that porcine kidney epithelial cells isolated from F1-generation gene-edited pigs could effectively inhibit TGEV infection. This study is the first to report the generation of APN-chimeric pigs, which may provide a natural host animal for characterizing PEC infection with APN and help in the development of better antiviral solutions.

Generation of PCBP1-deficient pigs using CRISPR/Cas9-mediated gene editing.

Classical swine fever virus (CSFV), a classic swine fever pathogen, causes severe economic losses worldwide. Poly (rC)-binding protein 1 (PCBP1), which interacts with Npro of CSFV, plays a vital role in CSFV growth. We are the first to report the generation of PCBP1-deficient pigs via gene-editing technology. The PCBP1-deficient pigs exhibited normal birth weight and reproductive-performance traits and developed normally. Viral challenge experiments indicated that primary cells isolated from F0- and F1-generation pigs exhibited significantly reduced CSFV infection. Additional mechanistic exploration further confirmed that the PCBP1 deficiency-mediated antiviral effect is related to the activation of type I interferon (IFN). Besides showing that a gene-editing strategy could be used to generate PCBP1-deficient pigs, our study introduces a valuable animal model for further investigating the infection mechanisms of CSFV that will help to develop better antiviral solutions.

Porcine ZC3H11A Is Essential for the Proliferation of Pseudorabies Virus and Porcine Circovirus 2.

Porcine epidemic viruses, such as pseudorabies virus (PRV) and porcine circovirus 2 (PCV2), are among the most economically damaging pathogens affecting the swine industry. Importantly, previous studies have shown that cases of human infection with PRV occur frequently, indicating the considerable risk of PRV transmission from pigs to humans. Zinc finger CCCH-type containing 11A (ZC3H11A) has been confirmed to play a crucial role in maintaining the nuclear export of mRNA under stress in humans, but its role in pigs remains unknown. In this study, we observed that ZC3H11A interacted with the transcription and export complex and played an important role in mRNA export. Specifically, we knocked out ZC3H11A in PK-15 cells with CRISPR/Cas9 and challenged them with PRV and PCV2. The results showed that the proliferation of the virus was significantly inhibited in ZC3H11A-/- cells, indicating that porcine ZC3H11A is indispensable for the proliferation of PRV and PCV2. Furthermore, our study demonstrated that the inactivation of ZC3H11A in host cells also inhibited the proliferation of PRV and PCV2. Taken together, the results of our study indicated that ZC3H11A is important for maintaining the export of mRNAs, which in turn facilitates the proliferation of PRV and PCV2, suggesting that it can be a potential target for producing antiviral pigs and drugs.

Identification and Functional Analysis of the Regulatory Elements in the pHSPA6 Promoter.

Functional and expressional research of heat shock protein A6 (HSPA6) suggests that the gene is of great value for neurodegenerative diseases, biosensors, cancer, etc. Based on the important value of pigs in agriculture and biomedicine and to advance knowledge of this little-studied HSPA member, the stress-sensitive sites in porcine HSPA6 (pHSPA6) were investigated following different stresses. Here, two heat shock elements (HSEs) and a conserved region (CR) were identified in the pHSPA6 promoter by a CRISPR/Cas9-mediated precise gene editing strategy. Gene expression data showed that sequence disruption of these regions could significantly reduce the expression of pHSPA6 under heat stress. Stimulation studies indicated that these regions responded not only to heat stress but also to copper sulfate, MG132, and curcumin. Further mechanism studies showed that downregulated pHSPA6 could significantly affect some important members of the HSP family that are involved in HSP40, HSP70, and HSP90. Overall, our results provide a new approach for investigating gene expression and regulation that may contribute to gene regulatory mechanisms, drug target selection, and breeding stock selection.

Generation of a pHSPA6 gene-based multifunctional live cell sensor.

Biosensors utilizing intact live cells can report responses to certain stimuli rapidly and sensitively and have attracted a great deal of attention. The expression pattern of HSPA6, a little studied HSPA family member, has contributed to the development of multifunctional and intelligent whole-cell sensors. Herein, a new pHSPA6-based EGFP fluorescent reporter cell line was designed and developed via a CRISPR/Cas9-mediated knock-in strategy. The fluorescent reporter cell line has a precise EGFP integration site and gene copy number, and no selectable marker genes were introduced during the selection processes. Stimulation experiments with HSPA6-specific stressors indicated that EGFP fluorescent reporter cells could rapidly and effectively convert stress signals into EGFP fluorescent signals. Furthermore, cell proliferation and gene expression pattern analysis showed that the fluorescent reporter cells grew well and that both the integrated EGFP gene and the pHSPA6 gene were expressed rapidly and sensitively in response to stimulation. This study provides a new strategy for the construction of a cell model for HSPA6 expression/interaction and an intelligent live cell sensor, which can potentially be applied to numerous fields, such as those focusing on cellular models of HSPA6 signaling cascades, biomaterials, food security, environmental assessment, and drug screening.

Generation of pRSAD2 gene knock-in pig via CRISPR/Cas9 technology.

A wide range of endemic and epidemic viruses, including classic swine fever virus (CSFV), pseudorabies virus (PRV) and others, are among the most economically important pathogens in pigs and have severely affected the national economy, human health and animal welfare and productivity. The RSAD2 exhibits antiviral activity against various DNA and RNA viruses. In this study, we successfully accomplished site-specific insertion of the porcine RSAD2 gene (pRSAD2) at the porcine ROSA26 (pROSA26) locus, generating pRSAD2 gene knock-in (pRSAD2-KI) PK-15 cells and porcine foetal fibroblasts (PFFs) via CRISPR/Cas9 technology. Gene expression analysis confirmed that pRSAD2-KI cells stably and efficiently overexpressed the pRSAD2 gene. Furthermore, viral challenge studies in vitro indicated that site-specific integration of the pRSAD2 gene not only effectively reduced CSFV infection but also PRV infection. More importantly, we ultimately successfully produced a pRSAD2-KI pig that constitutively overexpressed the pRSAD2, viral challenge results indicated that fibroblasts isolated from the pRSAD2-KI pig reduced CSFV infection. Taken together, these results suggest that CRISPR/Cas9-mediated knock-in strategy can be used for producing pRSAD2-KI pigs.