Porcine β-defensin 2 inhibits proliferation of pseudorabies virus in vitro and in transgenic mice

Abstract

Background: Porcine β-defensin 2 (PBD-2), produced by host cells, is an antimicrobial cysteine-rich cationic peptide with multi-functions. Previous studies have demonstrated that PBD-2 can kill various bacteria, regulate host immune responses and promote growth of piglets. However, the antiviral role of PBD-2 is rarely investigated. This study aimed to reveal the antiviral ability of PBD-2 against pseudorabies virus (PRV), the causative pathogen of Aujeszky's disease, in PK-15 cells and in a PBD-2 expressing transgenic (TG) mouse model. Methods: In this study, the cytotoxicity of PBD-2 on PK-15 cells was measured by CCK-8 assay. PK-15 cells were incubated with PRV pre-treated with different concentrations of PBD-2 and PRV titers in cell culture supernatants were determined by real-time quantitative PCR (RT-qPCR). TG mice and wild-type (WT) mice were intraperitoneally injected with PRV and the survival rate was recorded for 10 days. Meanwhile, tissue lesions in brain, spleen and liver of infected mice were observed and the viral loads of PRV in brain, liver and lung were analyzed by RT-qPCR.Results: PBD-2 at a maximum concentration of 80 μg/mL displayed no significant cytotoxicity on PK-15 cells. A threshold concentration of PBD-2 at 40 μg/mL was required to inhibit PRV proliferation in PK-15 cells. The survival rate in PBD-2 TG mice was 50% higher than that of WT mice. In addition, TG mice showed alleviated tissue lesions in brain, spleen and liver compared with their WT littermates after PRV challenge, while viral loads of PRV in brain, liver and lung of TG mice were significantly lower than that of WT mice.Conclusions: PBD-2 could inhibit PRV proliferation in PK-15 cells and protect mice from PRV infection, which confirmed the antiviral ability of PBD-2 both in vitro and in vivo. The application of PBD-2 in developing anti-viral drugs or disease-resistant animals can be further investigated.

Generation of Marker-Free pbd-2 Knock-in Pigs Using the CRISPR/Cas9 and Cre/loxP Systems

“…As a major group of cationic peptides widely existing in animals, defensins have been proven to possess broad antimicrobial activities against different pathogens including bacteria, fungi and viruses. Our previous in vivo studies showed that pigs overexpressing PBD-2 displayed increased resistance to A. pleuoropneumoniae infection [ 16 ], while mice expressing PBD-2 became more resistant to pseudorabies virus and Salmonella Typhimurium infections [ 27 , 28 ]. Given that the previous pbd-2 TG pigs were produced using random gene insertion, which also introduced a SMG, these would raise public concern on the biosafety of TG animals and hamper the commercialization of TG animals derived products.…”

“…The antibacterial mechanism of PBD-2 was elucidated in Escherichia coli , being disruption of the membrane integrity and affecting the DNA transcription and translation when PBD-2 entered into the cytoplasm [ 25 ]. Previous studies described that PBD-2 could hamper PRRSV replication in vitro [ 26 ] and suppress proliferation of pseudorabies virus both in vitro and in TG mice [ 27 ]. Besides, PBD-2 has been identified to alleviated inflammation by binding toll-like receptor 4 and inhibiting the subsequent NF-κB activation [ 28 ].…”

Pigs Overexpressing Porcine β-Defensin 2 Display Increased Resilience to Glaesserella parasuis Infection

“…Examples also include the insertion of an extra copy of the porcine CD28 gene into the genome, which improved the protective immune responses to PRRSV infection in pigs [ 62 ]. In addition to the described antibacterial activities of PBD-2, previous studies have demonstrated that PBD-2 could inhibit the proliferation of two major swine viruses, PRRSV and PRV, in different cell models [ 6 , 17 ]. These prompt us to investigate whether PBD-2 TG pigs can resist swine viral pathogen infections in the future.…”

“…Another study revealed that PBD-2 could provide protection against S. Typhimurium infection in TG mice through direct bactericidal ability and the inactivation of the TLR4/NF-κB pathway [ 16 ]. In addition, mice expressing PBD-2 became more resistant to pseudorabies virus (PRV) infection [ 17 ], while pigs overexpressing PBD-2 obtained improved resilience to Actinobacillus pleuropneumoniae infection [ 18 ]. Additionally, fibroblasts from our recently produced PBD-2 TG pigs displayed enhanced resistance to both Streptococcus suis and A. pleuropneumoniae infections [ 19 ].…”

The Antiviral Activity of Kaempferol Against Pseudorabies Virus in Mice

“…After mice were infected with PRV, histopathological analysis found that the brain, spleen, and liver tissues had different degrees of lesions [45]. Studies have shown that PRV could cause lethal respiratory disease in an animal model of PRV-infected BALB/c mice [46].…”