Nucleocytoplasmic Shuttling of Porcine Parvovirus NS1 Protein Mediated by the CRM1 Nuclear Export Pathway and the Importin α/ß Nuclear Import Pathway.

Porcine parvovirus (PPV) NS1, the major nonstructural protein of this virus, plays an important role in PPV replication. We show, for the first time, that NS1 dynamically shuttles between the nucleus and cytoplasm, although its subcellular localization is predominantly nuclear. NS1 contains two nuclear export signals (NESs) at amino acids 283 to 291 (designated NES2) and amino acids 602 to 608 (designated NES1). NES1 and NES2 are both functional and transferable NESs, and their nuclear export activity is blocked by leptomycin B (LMB), suggesting that the export of NS1 from the nucleus is dependent upon the chromosome region maintenance 1 (CRM1) pathway. Deletion and site-directed mutational analyses showed that NS1 contains a bipartite nuclear localization signal (NLS) at amino acids 256 to 274. Coimmunoprecipitation assays showed that NS1 interacts with importins α5 and α7 through its NLS. The overexpression of CRM1 and importins α5 and α7 significantly promoted PPV replication, whereas the inhibition of CRM1- and importin α/ß-mediated transport by specific inhibitors (LMB, importazole, and ivermectin) clearly blocked PPV replication. The mutant viruses with deletions of the NESs or NLS motif of NS1 by using reverse genetics could not be rescued, suggesting that the NESs and NLS are essential for PPV replication. Collectively, these findings suggest that NS1 shuttles between the nucleus and cytoplasm, mediated by its functional NESs and NLS, via the CRM1-dependent nuclear export pathway and the importin α/ß-mediated nuclear import pathway, and PPV proliferation was inhibited by blocking NS1 nuclear import or export. IMPORTANCE PPV replicates in the nucleus, and the nuclear envelope is a barrier to its entry into and egress from the nucleus. PPV NS1 is a nucleus-targeting protein that is important for viral DNA replication. Because the NS1 molecule is large (>50 kDa), it cannot pass through the nuclear pore complex by diffusion alone and requires specific transport receptors to permit its nucleocytoplasmic shuttling. In this study, the two functional NESs in the NS1 protein were identified, and their dependence on the CRM1 pathway for nuclear export was demonstrated. The nuclear import of NS1 utilizes importins α5 and α7 in the importin α/ß nuclear import pathway.

Two novel protein chips for the detection of antibodies against porcine parvovirus.

BACKGROUND: PPV is one of the most important pathogens causing porcine reproductive disorder. It has been shown in clinical cases to be a commonly mixed infection with other important swine diseases which can aggravate the severity of the disease and bring serious economic losses to the pig industry. Serological methods, such as hemagglutination inhibition assays (HAI), serum neutralization (SN), and the modified direct complement-fixation (MDCF) test were utilized earlier, whereas the enzyme-linked immunosorbent assay (ELISA) is the most frequently applied assay to detect PPV-specific antibodies. RESULTS: We establish the visible protein chip and the cyanine dye 3 (Cy3)-labeled protein chip to detect the clinical serum from pigs. In this study, the recombinant protein VP2 of PPV was expressed in E.coli, purified with nickel magnetic beads, and then printed onto epoxy-coated glass slides for preparation of the protein chip. After a series of experiments, the conditions of antigen protein concentration, incubation time of primary antibody or secondary antibody, and optimal serum dilution fold were optimized, resulting in a successful visible protein chip and Cy3-labeled protein chip. The results showed that the positive serum, diluted up to 6000-fold, can be detected by the visible protein chip, and the positive serum, diluted up to 12,800-fold, can be detected by the Cy3-labeled protein chip, suggesting the high sensitivity of these protein chips. Moreover, the positive detection ratio, sensitivity, and specificity of these two kinds of protein chips were higher than those of commercial ELISA antibody detection kits. CONCLUSION: Overall, these two protein chips can be used to rapidly diagnose clinical samples with high throughput.

Possible risks posed by single-stranded DNA viruses of pigs associated with xenotransplantation.

Routine large-scale xenotransplantation from pigs to humans is getting closer to clinical reality owing to several state-of-the-art technologies, especially the ability to rapidly engineer genetically defined pigs. However, using pig organs in humans poses risks including unwanted cross-species transfer of viruses and adaption of these pig viruses to the human organ recipient. Recent developments in the field of virology, including the advent of metagenomic techniques to characterize entire viromes, have led to the identification of a plethora of viruses in many niches. Single-stranded DNA (ssDNA) viruses are the largest group prevalent in virome studies in mammals. Specifically, the ssDNA viral genomes are characterized by a high rate of nucleotide substitution, which confers a proclivity to adapt to new hosts and cross-species barriers. Pig-associated ssDNA viruses include torque teno sus viruses (TTSuV) in the Anelloviridae family, porcine parvoviruses (PPV), and porcine bocaviruses (PBoV) both in the family of Parvoviridae, and porcine circoviruses (PCV) in the Circoviridae family, some of which have been confirmed to be pathogenic to pigs. The risks of these viruses for the human recipient during xenotransplantation procedures are relatively unknown. Based on the scant knowledge available on the prevalence, predilection, and pathogenicity of pig-associated ssDNA viruses, careful screening and monitoring are required. In the case of positive identification, risk assessments and strategies to eliminate these viruses in xenotransplantation pig stock may be needed.

Efficacy of inactivation of viral contaminants in hyperimmune horse plasma against botulinum toxin by low pH alone and combined with pepsin digestion.

Assuring viral safety of horse plasma-derived products is fundamental for ethical and regulatory reasons. We previously demonstrated the ability of pepsin digestion at low pH to inactivate West Nile and Sindbis viruses in horse plasma. The present study further examined the efficiency of pepsin digestion to inactivate four additional viruses: HSV-1 and BVDV (lipid-enveloped), BPV and Reo-3 (nonenveloped). These viruses were spiked into hyperimmunized horse plasma against botulinum toxin and subjected to low pH (3.2) alone or combined with pepsin digestion (1200 units/ml). Peptic digestion inactivated the lipid-enveloped viruses, whereas the nonenveloped viruses were unaffected. Interestingly, HSV-1 was rapidly inactivated by acidic pH alone (≥4.9 ± 0.6 log10), whereas a non-robust but meaningful BVDV inactivation (2.9 ± 0.7 log10) was achieved by combined low pH and pepsin. The current study demonstrated the ability of low pH alone and in combination with pepsin digestion to inactivate enveloped viral contaminants in anti-toxin horse plasma.

Codon Usage for Genetic Diversity, and Evolutionary Dynamics of Novel Porcine Parvoviruses 2 through 7 (PPV2-PPV7).

Porcine parvovirus (PPV) is the main pathogen of reproductive disorders. In recent years, a new type of porcine parvovirus has been discovered and named porcine parvovirus 2 to 7 (PPV2-PPV7), and it is associated with porcine circovirus type 2 in pigs. Codon usage patterns and their effects on the evolution and host adaptation of different PPV sub-types are still largely unknown. Here, we define six main sub-types based on the Bayesian method of structural proteins of each sub-type of PPV, including PPV2, PPV3, PPV4, PPV5, PPV6, and PPV7, which show different degrees of codon usage preferences. The effective number of codons (ENC) indicates that all PPV sub-types have low codon bias. According to the codon adaptation index (CAI), PPV3 and PPV7 have the highest similarity with the host, which is related to the main popular tendency of the host in the field; according to the frequency of optimal codons (FOP), PPV7 has the highest frequency of optimal codons, indicating the most frequently used codons in its genes; and according to the relative codon deoptimization index (RCDI), PPV3 has a higher degree. Therefore, it is determined that mutational stress has a certain impact on the codon usage preference of PPV genes, and natural selection plays a very decisive and dominant role in the codon usage pattern. Our research provides a new perspective on the evolution of porcine parvovirus (PPV) and may help provide a new method for future research on the origin, evolutionary model, and host adaptation of PPV.

Integration of Microfluidics, Photonic Integrated Circuits and Data Acquisition and Analysis Methods in a Single Platform for the Detection of Swine Viral Diseases.

Viral diseases challenge the health and welfare of pigs and undermine the sustainability of swine farms. Their efficient control requires early and reliable diagnosis, highlighting the importance of Point of Care (POC) diagnostics in veterinary practice. The objective of this study was to validate a novel POC system that utilizes Photonic Integrated Circuits (PICs) and microfluidics to detect swine viral pathogens using oral fluids and Porcine Parvovirus (PPV) and Porcine Circovirus 2 (PCV-2) as proofs of concept. The sensitivity and specificity of the device were calculated for both viruses, and Receiver Operating Characteristic (ROC) curves were drawn. PPV had an Area Under Curve (AUC) value of 0.820 (95% CI: 0.760 to 0.880, p < 0.0001), and its optimal efficiency threshold of detection shifts was equal to 4.5 pm (68.6% sensitivity, 77.1% specificity and Limit of Detection (LOD) value 106 viral copies/mL). PCV-2 had an AUC value of 0.742 (95% CI: 0.670 to 0.815, p < 0.0001) and an optimal efficiency threshold of shifts equal to 6.5 pm (69.5% sensitivity, 70.3% specificity and LOD 3.3 × 105 copies/mL). In this work, it was proven that PICs can be exploited for the detection of swine viral diseases. The novel device can be directly deployed on farms as a POC diagnostics tool.