Characterization of monoclonal antibodies that specifically differentiate field isolates from vaccine strains of classical swine fever virus.

Classical swine fever virus (CSFV) is a major animal pathogen threatening the global pork industry. To date, numerous anti-CSFV monoclonal antibodies (mAbs) and their recognizing epitopes have been reported. However, few mAbs were systematically characterized for the capacity to differentiate field CSFV isolates from CSF vaccine strains, and the molecular basis associated with antigenic differences between vaccines and field isolates is still largely unknown. In the present study, recombinant CSFV structural glycoproteins E2 of both virulent and vaccine strains and Erns of vaccine strain were expressed using eukaryotic cells and murine mAbs generated against E2 and Erns. After serial screening and cloning of the hybridomas, the viral spectra of mAbs were respectively determined by indirect fluorescent antibody assay (IFA) using 108 CSFVs, followed by Western blot analysis using expressed glycoproteins of all CSFV sub-genotypes including vaccine strains. The antigenic structures recognized by these mAbs were characterized by epitope mapping using truncated, chimeric, and site-directed mutated E2 and Erns proteins. We have identified two vaccine-specific, one field isolate-specific, and two universal CSFV-specific mAbs and five novel conformational epitopes with critical amino acid (aa) motifs that are associated with these five mAbs: 213EPD215, 271RXGP274, and 37LXLNDG42 on E2 and 38CKGVP42, W81, and D100/V107 on Erns. Particularly, E213 of E2 is field isolate-specific, while N40 of E2 and D100/V107 of Erns are vaccine strain-specific. Results from our study further indicate that N40D of E2 mutation in field strains was likely produced under positive selection associated with long-term mass vaccination, leading to CSFV evasion of host immune response. Taking together, this study provides new insights into the antigenic structure of CSFV E2 and Erns and the differentiating mAbs will contribute to the development of a diagnostic strategy to differentiate C-strain vaccination from natural infection (DIVA) of CSFV in terms of elimination of CSF in China.

A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses.

Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections.

Detection of bovine central nervous system tissue as bovine spongiform encephalopathy risk material by real-time reverse transcriptase-PCR in raw and cooked beef products.

There is increasing evidence of the association of the new variant of Creutzfeldt-Jacob disease (nvCJD) in humans with bovine spongiform encephalopathy (BSE) in cattle. Many countries established legislation of banning central nervous system (CNS) tissues, which are regarded as BSE-specified risk materials (SRM), in human food supply because of the potential transmission of BSE to humans. A real-time reverse transcriptase-PCR assay using the bovine glial fibrillary acidic protein (GFAP) mRNA template for the detection of CNS tissues in raw and cooked beef products was developed in this study. The results showed that (1) this method can detect CNS tissues from bovine and ovine origins, but not from porcine and avian ones; (2) GFAP mRNA can only be detected from brain and spinal cords rather than other tissues; (3) the GFAP mRNA was detectable in CNS tissues even after dilution to 0.001%; and (4) the assay was unaffected by heat treatment at 100 degrees C for 30 min or storage at room temperature for 4 days, and at 4 degrees C for at least 15 days.

Immune enhancing effects of recombinant bovine IL-18 on foot-and-mouth disease vaccination in mice model.

Foot-and-mouth disease (FMD) is a highly contagious disease in cloven-hoofed animals and can cause a considerable socio-economic loss for affected countries. Interleukin-18 (IL-18) is a pleiotropic cytokine and plays important role in both the development of a functional immune system as well as the response of the organism to infection. In the present study, bovine IL-18 (BoIL-18), Foot-and-mouth disease virus VP1 and VP1/BoIL-18 fusion genes were cloned and expressed in pichia pastoris (P. pastoris) and subsequently immune effects were evaluated to study the immune enhancing effects of recombinant BoIL-18 (rBoIL-18) on FMD vaccination. The results showed that the genes encoding for BoIL-18, VP1 and VP1/BoIL-18 are successfully expressed in P. pastoris and the expressed recombinant VP1 (rVP1) proteins could induce both humoral and marginal cell-mediated immune responses in mice, while the co-inoculation with rBoIL-18 could markedly enhance both of immune responses, and the inoculation of the fusion product rVP1/BoIL-18 showed even more dramatic immune responses, suggesting rBoIL-18 has a potential to enhance the efficacy of vaccination against FMDV infection.

Interferon-alpha genes from Bos and Bubalus bubalus.

Interferon-a genes were cloned from six breeds of three species of two genera (three Chinese native cattle breeds of yellow cattle, wild yak and HuanHu domestic yak, one European breed of Holstein cow, and two water buffalo breeds of FuAn water buffalo and FuZhong water buffalo) by direct PCR. The PCR products were directly inserted into the expression vector to be sequenced and expressed. Sequence analysis showed that IFN-a genes of six clones were composed of 498 nucleotides, encoding a mature polypeptide with 166 amino acids. Compared with the published BoIFN-a subtypes, the IFN-a gene of Holstein cow had only one point mutation with the BoIFN-aA subtype. The IFN-a gene of yellow cattle was similar to the BoIFN-aD subtype with amino acid identity of 97.0% and may be considered as a new subtype, namely, BoIFN-aD1. The other four IFN-a genes, cloned from wild yak and HuanHu domestic yak, FuAn water buffalo, and FuZhong water buffalo, represented four new subtypes, namely, BoIFN-aI, BoIFN-aJ, BuIFN-a1, and BuIFN-a2, respectively. Each of the six clones was expressed in E. coli with molecular weight of approximately 20 kDa by SDS-PAGE and Western blot analyses. Antiviral activity assays showed that the six recombinant IFN-a (rIFN-a) all exhibited 1,000 times higher antiviral activity in the MDBK/VSV cell line than in the CEF/VSV one. Moreover, the rIFN-as could inhibit infectious bovine rhinotracheitis virus replication in the MDBK cell line using CPE inhibition method. The results suggested that rIFN-as a potential agent for clinical application against virus diseases in cattle industry.

Expressions of Bovine IFN-gamma and foot-and-mouth disease VP1 antigen in P. pastoris and their effects on mouse immune response to FMD antigens.

As a highly contagious disease in cloven-hoofed animals, foot-and-mouth disease virus (FMDV) may cause a considerable social-economic loss in those countries affected. IFN-gamma has a wide range of antiviral and immune modulating functions. Thus, to study the immune enhancing effects of recombinant Bovine IFN-gamma (rBoIFN-gamma) on a recombinant FMDV vaccine, BoIFN-gamma, FMDV VP1 and BoIFN-gamma/VP fusion genes were cloned, expressed, co-expressed in pichia pastoris (P. pastoris) respectively, and subsequent immune effects have been evaluated in this study. The results showed that the genes encoding for BoIFN-gamma, VP1 and BoIFN-gamma/VP1 are successfully expressed in P. pastoris and their products are directly secreted into the cultural supernatant at a high level of 1.0 g/L analyzed by thin-layer scanning. In addition, rVP1 alone could induce both humoral and marginal cell-mediated immune responses in mice, while the group with co-inoculations of rBoIFN-gamma could markedly enhance both humoral and cell-mediated immune responses; even more dramatic immune responses were observed with the group inoculated with the fusion product, rBoIFN-gamma/VP1. The fusion product could be further investigated for its utility of FMDV vaccine development.