A poly(dimethylsiloxane)-based solid-phase microchip platform for dual detection of Pseudorabies virus gD and gE antibodies.

Pseudorabies caused by pseudorabies virus (PRV) infection is still a major disease affecting the pig industry; its eradication depends on effective vaccination and antibody (Ab) detection. For a more rapid and accurate PRV detection method that is suitable for clinical application, here, we established a poly(dimethylsiloxane)-based (efficient removal of non-specific binding) solid-phase protein chip platform (blocking ELISA) for dual detection of PRV gD and gE Abs. The purified gD and gE proteins expressed in baculovirus were coated into the highly hydrophobic nanomembrane by an automatic spotter, and the gray values measured by a scanner were used for the S/N (sample/negative) value calculation (gD and gE Abs standard, positive: S/N value ≤0.6; negative: S/N value >0.7; suspicious: 0.6 < S/N ≤ 0.7). The method showed an equal sensitivity in the gD Ab test of immunized pig serum samples compared to the neutralization test and higher sensitivity in the gE Ab test compared to the commercial gE Ab detection kit. In the clinical evaluation, we found an agreement of 100% (122/122) in the gD Ab detection compared to the neutralization test and an agreement of 97.5% (119/122) in the gE Ab detection compared to the commercial PRV gE Ab detection kit. In summary, the protein chip platform for dual detection of PRV gD and gE Abs showed high sensitivity and specificity, which is suitable for PRV immune efficacy evaluation and epidemic monitoring.

Identification of a conserved linear B-cell epitope at the N-terminus of the E2 glycoprotein of Classical swine fever virus by phage-displayed random peptide library.

The E2 protein of Classical swine fever virus (CSFV) is an important envelope glycoprotein, which is responsible for inducing protective immune response in swine. Four antigenic domains, A-D, have been mapped on the E2 protein. The present study describes the identification of a linear B-cell epitope at the N-terminus of the E2 protein by screening a phage-displayed random 12-peptide library with the neutralizing monoclonal antibody (mAb) HQ06 directed against the E2 protein. HQ06 was found to bind to the phages displaying a consensus motif LFDGSNP, which is highly homologous to (772)LFDGTNP(778) of the CSFV polyprotein, locating on the border between antigenic domains B/C and A of the E2 protein. Considering that HQ06 showed reactivity with the motif (772)LFDGTNP(778) expressed as GST fusion in Western blot and indirect ELISA, we propose that the motif (772)LFDGTNP(778) represents a linear B-cell epitope of the E2 protein. The motif (773)FDGTNP(778) is the minimal requirement for the reactivity as demonstrated by analysis of the reactivity of HQ06 with several truncated peptides derived from the motif. Alignment of amino acid sequences from a number of CSFV isolates indicated that the epitope is well conserved among different subgroups of CSFV. Substitutions of the individual residues within the epitope (773)FDGTNP(778) demonstrated that residues (773)F, (775)G, and (778)P constitute the core of the epitope. The identified epitope will be useful for development of diagnostic assays and epitope-based marker vaccines against CSFV.

[Differences in glycosylation of the E2 protein between virulent Shimen strain and a virulent C-strain of classical swine fever virus].

The E2 envelope glycoprotein of virulent Shimen strain and avirulent C-strain of Classical swine fever virus (CSFV) has 5 and 6 potential glycosylation sites, respectively, and the potential glycosylation site 986N is unique to C-strain. To study the differences in glycosylation between the virus pair, the E2 genes (removing signal sequence and transmembrane anchor regions) of the two strains fused with the melittin signal sequence were expressed in the Sf9 insect cells. The recombinant E2 proteins were secreted into the medium of Sf9 cells in dimer form with different molecular weight (MW). Deglycosylation of the recombinant E2 proteins by endo H and PNGase F showed that the deglycosalated Shimen-E2 and HCLV-E2 have the same MW, indicating that the different MW between Shimen-E2 and HCLV-E2 proteins came from different glycosylation. Site-directed mutagenesis in the potential glycosylation site at 986N demonstrated that the mutated Shimen-E2 protein had the same MW as the wild-type HCLV-E2 protein, while the mutated HCLV-E2 had the same MW as the wild-type Shimen-E2 protein. We suggest that the different MW between Shimen-E2 and HCLV-E2 is resulted from the different glycosylation on 986 N glycosylation site.

Efficient gene delivery into mammalian cells mediated by a recombinant baculovirus containing a whispovirus ie1 promoter, a novel shuttle promoter between insect cells and mammalian cells.

Recombinant baculoviruses have emerged as a new gene delivery vehicle for mammalian cells. Thus, a shuttle promoter that mediates gene expression in both insect and mammalian cells will facilitate the development of a baculovirus vector-based mammalian cell gene delivery vehicle. This study described the generation of three recombinant baculoviruses with an EGFP reporter gene under the control of the white spot syndrome virus (WSSV) ie1 promoter, or either of two control promoters, the baculovirus early-to-late (ETL) promoter and polyhedrin promoter. The resulting recombinant baculoviruses were used to infect insect Sf9 cells and transduce several mammalian cell lines to test the expression of EGFP. We found that the WSSV ie1 promoter displayed a strong promoter activity in both insect and mammalian cells, and showed a stronger promoter activity than the ETL promoter in some mammalian cell lines. The activity of the WSSV ie1 promoter, but not the ETL promoter, can be enhanced by sodium butyrate, a histone deacetylase inhibitor. A transient plasmid transfection assay indicated that the WSSV ie1 promoter activity in mammalian cells is independent of baculovirus gene expression, differing from the ETL promoter, which was shown to be baculovirus-dependent. This study demonstrates, for the first time, that the WSSV ie1 promoter can function as a baculovirus-independent shuttle promoter between insect cells and mammalian cells. This novel shuttle promoter will facilitate the application of baculovirus-based vectors in gene expression, gene therapy, and non-replicative vector vaccines.

A Semliki Forest virus replicon vectored DNA vaccine expressing the E2 glycoprotein of classical swine fever virus protects pigs from lethal challenge.

Classical swine fever virus (CSFV) causes significant losses in pig industry in many countries in Asia and Europe. The E2 glycoprotein of CSFV is the main target for neutralizing antibodies. Recently, the replicon of alphaviruses, such as Semliki Forest virus (SFV), has been developed as replicative expression vectors for gene delivery. In this study, we constructed a plasmid DNA based on SFV replicon encoding the E2 glycoprotein of CSFV and evaluated its efficacy in rabbits and pigs. The results showed that the animals immunized with the DNA vaccine developed CSFV-specific neutralizing antibodies and were protected from virulent or lethal challenge. This demonstrates that the SFV replicon-derived DNA vaccine can be a potential marker vaccine against CSFV infections.