Wnt/ß-catenin signaling pathway inhibits porcine reproductive and respiratory syndrome virus replication by enhancing the nuclear factor-κB-dependent innate immune response.

The Wnt/ß-catenin signaling pathway is an evolutionarily highly conserved signaling pathway related to the replication of various viruses. However, the interaction between the Wnt/ß-catenin pathway and porcine reproductive and respiratory syndrome virus (PRRSV) is unknown. In the present study, we showed that PRRSV-infected Marc-145 and PAM cells expressed high levels of c-myc and cyclinD1 mRNA and accumulation of ß-catenin in the nucleus. PRRSV nonstructural proteins (Nsps) 1α, 1ß, 3, 4, 7, 10, and 12, and proteins encoded by open reading frames (ORFs) 2b, 3, and 5 induced the activation of the Wnt pathway according to TOP/FOP luciferase reporter assay. But, Nsp5 inhibited the activation of the Wnt pathway. Pre-treatment with Wnt3a inhibited PRRSV replication in Marc-145 cells in a dose-dependent manner. Over-expression of ß-catenin also inhibited PRRSV replication, while silencing of ß-catenin by small hairpin RNA increased its replication in Marc-145 cells. Over-expression of ß-catenin increased interferon regulatory factor (IRF)-3 expression and nuclear factor (NF)-κB phosphorylation, NF-κB and interferon-stimulated response element promoter activities, and interferon-ß, DExD/H-box helicase 58 (DDX58), interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-8 mRNA expression. Conversely, silencing ß-catenin decreased phosphorylated IRF-3 and NF-κB, NF-κB and IFIT1 promoter activities, and IFN-ß, DDX58, IFIT1, IL-1ß, TNF-α, and IL-8 mRNA levels in Marc-145 cells. Co-immunoprecipitation and immunofluorescence colocalization analyses confirmed that ß-catenin interacted with NF-κB in Marc-145 cells. In conclusion, PRRSV infection activates the Wnt/ß-catenin signaling pathway via Nsps 1α, 1ß, 3, 4, 7, 10, and 12, and proteins encoded by ORFs 2b, 3, and 5. The Wnt/ß-catenin pathway then inhibits PRRSV replication by enhancing the NF-κB-dependent innate immune response. These findings further our understanding of the role of the Wnt/ß-catenin signaling pathway in regulating PRRSV replication and provide new insights into virus-host interactions.

Development of a Dual Fluorescent Microsphere Immunological Assay for Detection of Pseudorabies Virus gE and gB IgG Antibodies.

Pseudorabies, also known as Aujezsky's disease, is an acute viral infection caused by pseudorabies virus (PRV). Swine are one of the natural hosts of pseudorabies and the disease causes huge economic losses in the pig industry. The establishment of a differential diagnosis technique that can distinguish between wild-type infection and vaccinated responses and monitor vaccine-induced immunoglobulin G(IgG) is crucial for the eventual eradication of pseudorabies. The aim of this study was to develop a rapid dual detection method for PRV gE and gB protein IgG antibodies with high specificity and sensitivity. PRV gE codons at amino acid residues (aa) 52-238 and gB codons at aa 539-741 were expressed to obtain recombinant PRV gE and gB proteins via a pMAL-c5x vector. After purification with Qiagen Ni-nitrilotriacetic acid (NTA) agarose affinity chromatography, the two proteins were analyzed via SDS-PAGE and immunoblotting assays. Two single fluorescent-microsphere immunoassays (FMIAs) were established by coupling two recombinant proteins (gE and gB) to magnetic microbeads, and an effective dual FMIA was developed by integrating the two single assays. Optimal serum dilution for each assay, correlation with other common swine virus-positive sera, and comparison with ELISA for two PRV antigens were tested for validation. Compared with ELISA, the specificity and sensitivity were 99.26% and 92.3% for gE IgG antibody detection, and 95.74% and 96.3% for the gB IgG antibody detection via dual FMIA. We provide a new method for monitoring PRV protective antibodies in vaccinated pigs and differentiating wild-type PRV infection from vaccinated responses simultaneously.

First report and genetic diversity of porcine bufavirus in China.

BACKGROUND: Bufavirus is a newly discovered zoonotic virus reported in numerous mammals and humans. However, the epidemiological and genetic characteristics of porcine bufaviruses (PBuVs) in China remain unclear. METHODS: To detect PBuVs in China, 384 samples (92 fecal and 292 serum specimens) were collected from 2017 to 2018, covering six provinces in China, and were evaluated by nested PCR. Further, the positive samples from different provinces were selected to obtain the complete genome of Chinese PBuVs. RESULTS: The prevalence rate of PBuV was 16.7% in Chinese domestic pigs in the Guangdong, Guangxi, Fujian, Jiangxi, Anhui, and Henan provinces. Additionally, the positive rate of fecal specimens was higher than that of the serum samples. Next, we sequenced nine near-complete genomes of Chinese field PBuV strains from different provinces. Homology and phylogenetic analyses indicated that Chinese PBuVs have high genetic variation (93.3-99.2%), showed higher nucleotide identity with an Austrian PBuV strain (KU867071.1), and developed into different branches within the same cluster. CONCLUSION: To our knowledge, this is the first report on PBuV in China, expanding the geographic boundaries of PBuV circulation. Our data demonstrate that PBuVs are widely distributed in the six Chinese provinces. Moreover, these Chinese PBuVs exhibit genetic variation and continuous evolution characteristics. Taken together, our findings provide a foundation for future studies on bufaviruses.

Phages bearing specific peptides with affinity for porcine reproductive and respiratory syndrome virus GP4 protein prevent cell penetration of the virus.

Porcine reproductive and respiratory syndrome (PRRS) has caused significant economic losses to the pig industry worldwide over the last 30 years. GP4 is a minor highly glycosylated structural protein composed of 187 and 183 amino acids in types I and II porcine reproductive and respiratory syndrome virus (PRRSV), respectively. The GP4 protein co-localizes with cluster of differentiation 163 (CD163), the major receptor on the target cell membrane, to mediate PRRSV internalization and disassembly. However, it remains to be established whether blocking interactions between GP4 and host cells can inhibit viral proliferation. In the present study, recombinant GP4 protein prepared and purified using the Escherichia coli system effectively recognized PRRSV-positive serum. Phage display biopanning on GP4 protein showed that the specific phages obtained could distinguish PRRSV from the other viruses. The exogenous peptide WHEYPLVWLSGY displayed on one of the candidate phages showed high affinity for GP4 protein and exerted a significant inhibitory effect on PRRSV penetration in vitro. Moreover, the N-terminus of GP4 was predicted as the critical receptor binding site and the beginning of the fifth scavenger receptor cysteine-rich domain of CD163 as the critical ligand recognition site based on sequence alignment and model prediction analyses. The current study expands our understanding of PRRSV GP4 and its receptor CD163 and provides a fresh perspective for the development of novel peptide-based viral inhibition reagents.