Analysis of murine B-cell epitopes on bluetongue virus 12 nonstructural protein 1.

The bluetongue virus (BTV) NS1 protein is one of the major proteins synthesized during BTV infection and is responsible for the generation of virus-specific tubules. Although some functional and structural studies on the BTV NS1 protein have been reported, there have been no reports describing the linear B-cell epitopes recognized by humoral immune responses published to date. In this study, 25 BTV12 NS1-reactive monoclonal antibodies (MAbs) and polyclonal antisera (polyclonal antibodies, PAbs) were generated and analyzed. We identified 14 linear NS1 epitopes recognized by the PAbs and MAbs using NS1-derived peptides in an enzyme-linked immunosorbent assay. Moreover, we predicted 23 linear B-cell epitopes using the ABCpred online server which employs an artificial neural network. Analysis of the predicted and identified epitopes of NS1 demonstrated the feasibility of B-cell epitope prediction. Sequence alignments indicated that the epitopes recognized by MAbs are highly conserved among BTV serotypes, but not among the other members of the genus Orbivirus, such as the African horse sickness virus (AHSV), epizootic hemorrhagic disease virus (EHDV), and Chuzan disease virus (CV). Importantly, we identified specific MAbs that recognized all BTV serotypes tested as well as MAbs that recognized only BTV12, suggesting that these NS1-specific MAbs could serve as a basis for BTV diagnostic approaches. The generation and identification of NS1 protein epitopes will provide the foundation for further studies about the function and structure of NS1 and novel epitope-based vaccines.

Analysis of murine B-cell epitopes on Eastern equine encephalitis virus glycoprotein E2.

The Eastern equine encephalitis virus (EEEV) E2 protein is one of the main targets of the protective immune response against EEEV. Although some efforts have done to elaborate the structure and immune molecular basis of Alphaviruses E2 protein, the published data of EEEV E2 are limited. Preparation of EEEV E2 protein-specific antibodies and define MAbs-binding epitopes on E2 protein will be conductive to the antibody-based prophylactic and therapeutic and to the study on structure and function of EEEV E2 protein. In this study, 51 EEEV E2 protein-reactive monoclonal antibodies (MAbs) and antisera (polyclonal antibodies, PAbs) were prepared and characterized. By pepscan with MAbs and PAbs using enzyme-linked immunosorbent assay, we defined 18 murine linear B-cell epitopes. Seven peptide epitopes were recognized by both MAbs and PAbs, nine epitopes were only recognized by PAbs, and two epitopes were only recognized by MAbs. Among the epitopes recognized by MAbs, seven epitopes were found only in EEEV and two epitopes were found both in EEEV and Venezuelan equine encephalitis virus (VEEV). Four of the EEEV antigenic complex-specific epitopes were commonly held by EEEV subtypes I/II/III/IV (1-16aa, 248-259aa, 271-286aa, 321-336aa probably located in E2 domain A, domain B, domain C, domain C, respectively). The remaining three epitopes were EEEV type-specific epitopes: a subtype I-specific epitope at amino acids 108-119 (domain A), a subtype I/IV-specific epitope at amino acids 211-226 (domain B) and a subtype I/II/III-specific epitope at amino acids 231-246 (domain B). The two common epitopes of EEEV and VEEV were located at amino acids 131-146 and 241-256 (domain B). The generation of EEEV E2-specific MAbs with defined specificities and binding epitopes will inform the development of differential diagnostic approaches and structure study for EEEV and associated alphaviruses.

Measuring the expression of exogenous BDNF of NSCs after infection of supernatant from recombi-nant retrovirus pLXSN-BDNF / 中华微生物学和免疫学杂志

Objective To develop neural stem cells(NSCs) which can stably express exogenous brain-derived neurotrophic factor(BDNF) in vitro. Methods NSCs from the subependymal zone of embry-onic day 14.5(E14.5) rat brain were purified by limiting dilution assay and then infected with supernatant of recombinant retrovirus pLXSN-BDNF and retrovirus pLXSN. The original copy numbers of exogenous gene templates from three groups NSCs(pLXSN-BDNF viral infection group, pLXSN viral infection group, control group) were detected by fluorescent quantitative PCR(FQ-PCR). ELISA assay was used for determining the protein contents of BDNF of supernatant from three groups NSCs for six days continually after seeded in 24-well plates in the same cell density. Results NSCs were purified successfully by limiting dilution assay.The original copy numbers of exogenous BDNF gene templates from pLXSN-BDNF viral infection group by FQ-PCR were (19.57±0.65) × 10~3 copies/μl, higher than those of another two groups(P < 0.05). The protein contents of BDNF of supernatant from NSCs of pLXSN-BDNF viral infection group was highest among three groups and compared with another two groups had statistical significance (P <0.05) . Conclusion The purified NSCs can be transduced exogenous BDNF successfully with supematant of recombinant retrovir-us pLXSN-BDNF which provide experimental evidences and laying foundations for further research of retinal transplantation and quantization investigation of gene therapy for optic nerve injury.

Antiviral activity determination of recombinant equine interferon-gamma and identification inhibited antiviral activity of monoclonal antibodies / 生物工程学报

Equine interferon-gamma (eIFN-gamma) expressed both in E. coli and baculovirus were evaluated for antiviral activity against recombinant Vesicular Stomatits Virus expressing green fluorescence protein (rVSV-GFP) in EFK-78 cells. The assays were conducted in 96-well plate. Virus infectivity was measured by quantifying GFP-positive cells, instead of quantifying the CPE reduction. Prior to infection of EFK-78 cells with rVSV-GFP, the cells were incubated with eIFN-gamma. The GFP expression in the EFK-78 cells dramatically decreased in the cells treated with eIFN-gamma in a dose-dependent manner, comparing with the mock-treated cells. The titers of antiviral activity were 1 x 10(3) AU/mL and 1 x 10(5) AU/mL of eIFN-gamma expressed from E. coli and baculovirus, respectively. The antiviral activities of the recombinant eIFN-gamma were highly efficient and specific, as it was blocked by mAbs against eIFN-gamma.