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Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which causes great economic losses. At the moment, no effective neutralizing antibody is available for scientific research and treatment. Therefore, developing a method for screening the neutralizing monoclonal antibodies is of great significance for the prevention and treatment of PRRSV and the screening of antigen sites. Monoclonal antibodies have been widely used in the treatment and diagnosis of many human and animal diseases. Therefore, screening effective neutralizing antibodies for different pathogens is an urgent task. Among the methods for monoclonal antibody screening, B cell immortalization is an effective method to obtain neutralizing monoclonal antibody. Specifically, in this study, the bcl-6 and bcl-xl genes were connected by f2a and then the yielded product was ligated to a vector for retrovirus packaging. The swine lymphocytes immunized with PRRSV were infected the yielded mature viruses and cultured in the complete medium containing CD40L and IL21 cytokines. Then, CD21 was used as the marker to screen B cells with the magnetic bead method. Finally, monoclonal B cells were obtained and the secretion of antibodies was tested. The results showed that the plasmid, either being transfected alone or with the packaged plasmids, could be expressed, and that the packaged retrovirus could infect the cells. Moreover, the infected lymphocytes secreted antibodies, so did the screened B cells. Therefore, the method for screening monoclonal antibody against PRRSV was successfully established.

Antigenic purity is important for quality control of the foot-and-mouth (FMD) whole virus inactivated vaccine. The recommended method for evaluation the antigenic purity of FMD vaccine is to check the serum conversion to non-structural protein (NSP) 3AB antibody after 2 to 3 times inoculation of animals with inactivated vaccine. In this study, we developed a quantitative ELISA to detect the amount of residual 3AB in vaccine antigen, to provide a reference to evaluate the antigenic purity of FMD vaccine. Monoclonal antibody (Mab) of NSP 3A and HRP-conjugated Mab of NSP 3B were used to establish a sandwich ELISA to quantify the NSP 3AB in vaccine antigen of FMD. Purified NSP 3AB expressed in Escherichia coli was serially diluted and detected to draw the standard curve. The detectable limit was determined to be the lowest concentration of standard where the ratio of its OD value to OD blank well was not less than 2.0. Results: The OD value was linearly corelated with the concentration of 3AB protein within the range between 4.7 and 600 ng/mL. The correlation coefficient R² is greater than 0.99, and the lowest detectable limit is 4.7 ng/mL. The amount of 3AB protein in non-purified inactivated virus antigen was detected between 9.3 and 200 ng/mL depending on the 12 different virus strains, whereas the amount of 3AB in purified virus antigen was below the lowest detectable limit. The amount of 3AB in 9 batches of commercial FMD vaccine antigens was between 9.0 and 74 ng/mL, whereas it was below the detectable limit in other 24 batches of commercial vaccine antigens. Conclusion: the sandwich ELISA established in this study is specific and sensitive to detect the content of 3AB protein in vaccine antigen of FMD, which will be a useful method for evaluation of the antigenic purity and quality control of FMD inactivated vaccine.

Objective: A series of type O foot-and-mouth disease (FMD) outbreaks occurred in China seriously affect development of Chinese husbandry. Its causative agent-type O foot-and-mouth disease virus (FMDV) has been evolved three topotypes: Cathay, Middle East-South Asia and Southeast Asia, specifically, the viruses of Cathay topotype are highly adapted to pig, representing the biggest threat on Chinese pig industry. The available FMD vaccine in China provides insufficient protection against some arising viruses of Chathay topotype, which exert important obstacle to control porcinophilic FMD epidemics in China. To develop vaccine candidate with characteristics of good immunogenicity and broad spectrotype, a full-length infectious cDNA clone of inter-genotypic chimeric FMDV was constructed, which replaced part VP3 and VP1 gene of O/HN/93 strain with the corresponding to the variants of Cathay topotype (mainly replaced the B-C and G-H loop of structure protein VP1)., Methods: Linearized recombinant plasmid and plasmid expressing T7 RNA polymerase were cotransfected into BHK-21 cells to rescue the chimeric virus in vivo., Result: The transfected cells showed apparent cytopathic effects (CPE) after 36 h post-transfection. The rescued virus was checked by RT-PCR, indirect immunofluorescence, electron microscope. Results show that chimeric FMDV was successfully rescued in vivo. The study of sulk mice pathogenicity show chimeric FMDV has attenuated pathogenicity for suckling mice compared with its parental virus., Conclusion: The construction of inter-genotypic chimeric FMDV will lay the basis for developing novel vaccine against FMD.

We investigated the enhanced immune response of a recombinant T cell immunogen as an effective cellular immune adjuvant. The T cell immunogen named TI contained several T cell epitopes from the VP1, VP4, 3A and 3D proteins of foot-and-mouth disease virus (FMDV) and two pan-T helper (T(H)) cell sites to broaden the immunogenicity of the protein. Meanwhile, another fusion protein named OA-VP1 was expressed in bacteria, which contained two VP1 proteins of O and Asia1 type FMDV. Mice were vaccinated with commercially inactivated vaccine or OA-VP1 protein with or without the TI immunogen. The results show that mice inoculated with inactivated vaccine or OA-VP1 protein supplemented with TI immunogen produced significantly higher level of neutralizing antibodies (P < 0.01 or P < 0.05) than the mice only inoculated with inactivated vaccine or OA-VP1 protein by microneutralization assay. An obvious increase in T cell number by flow cytometric analysis and significantly higher concentration of IFN-gamma secreted in culture media of spleen lymphocytes were observed in groups supplemented with TI immunogen (P < 0.01). TI immunogen was an effective stimulator for humoral and cellular immunity and could help improve the immunogenicity of inactivated vaccine or protein subunit vaccine.

Objective: Foot-and-Mouth Disease Virus (FMDV) initiates infection by binding to integrin receptors via a highly conserved Arg-Gly-Asp (RGD) sequence found in the G-H loop of the structural protein VP1. However, FMDV is an RNA virus, which harbors the evolutionary potential to render the RGD motif dispensable upon changes in constant environment. We studied mutation of RGD motif upon short-time passages FMDV Asial/JS/China/2005 field strain in different host., Methods: VP1 gene was amplified from Asial/JS/China/05 field strain, the fourth passage virus in sulking mice (MF4) of the above strain and the virus isolated from a pig housing with cattle inoculated with the above strain followed eight passages in BHK-21 cell (PBF8) by RT-PCR, and the VP1 genes were sequenced and their deduced amino acid sequences were compared with each other., Result: Dominant population with RGD and Arg-Ser-Asp (RSD) receptor recognition site motif was generated after four passages of Asial/JS/China/2005 field virus in sulking mice and another dominant population with Arg-Asp-Asp (RDD) motif was produced after eight passages the virus isolated from housing pig with cattle inoculated with the above field strain., Conclusion: This study indicated that the dominant FMDV virus populations with RSD or RDD receptor binding site instead of original RGD motif were produced upon short-time evolution of FMDV field isolates with RGD motif in different environment. These studies not only increase number of viable mutants with substitutions in the RGD region, but also these profoundly altered, but viable, mutants with different receptor recognition site will provide useful tools for studies of cell recognition by FMDV and host tropism modifications.

Objective: To construct an infectious full-length cDNA clone of Asial/JS/China/2005 strain with Arg-Gly-Asp (RGD) receptor recognition site., Methods: We constructed foot-and-mouth disease virus type Asial full-length cDNA clone pFMDV-RGD by using site-directed mutagenesis. The plasmid pFMDV-RGD contained the desired mutation. The plasmids pFMDV-RGD were linearized with NotI enzyme. Linearized plasmid and pcDNAT7P plasmids expressing T7 RNA polymerase cotransfected into BHK-21 cells to rescue FMDV-RGD., Results: We constructed FMDV Asial/JS/China/2005 strain full-length cDNA clone with Arg-Gly-Asp receptor recognition site by sequence. We obtained rescued virus by plasmid cotransfection. The results of sequencing, indirect immunofluorescence, electron microscope and sulk mice pathogenicity analysis showed foot-and-mouth disease virus containing Arg-Gly-Asp receptor recognition site was successfully rescued., Conclusion: The results lay a foundation for further study of biology characteristic diversity of rescued virus with Arg-Gly-Asp and Arg-Asp-Asp (RDD) receptor recognition site.

Objective: Study the association of the length of poly(C) tract with virulence of foot-and-mouth disease virus., Methods: The recombinant plasmids pGEM-XJ/AKT/69 containing the full-length cDNA of FMDV were linearized by Nhe I /Not I and transcribed by T7 RNA polymerase. The in vitro transcripts were transfected into BHK-21 cells using Lipofectamine 2000 reagent. Then, the genetic engineering virus was rescued from BHK-21 cells. The poly(C) tracts were sequenced at different passages of rescued virus, and the pathogenicities were evaluated with 3-day-old mice and BHK-21 cells by detection of LD50 and TCID50., Results: After six passages in BHK-21 cells, cytopathic effect was observed by microscopy. The rescued virus was rejuvenated once in unweaned mice, and then came back to cell passage. We found that the poly(C) tract of rescued virus was shortened when the virus was passaged twice in BHK-21 cells. Although the data of LD50 in mice and TCID50 in BHK-21 cells showed that the virulence and infectivity of genetic engineering viruses were lower than its parental virus, no significant difference was observed between the genetic engineering viruses with the different length poly(C) tract., Conclusion: The length of poly(C) tract ranged from 12 to 17 nucleotides did not cause significant influence on the virulence and infectivity of genetic engineering virus.

Objective: To study the cell cycle block and apoptosis induced by hydroxycamptothecine (HCPT) in cultured rabbit lens epithelial cells (RLECs) and its affect on the gene expression of fas/fas-L in RLECs., Methods: Flow cytometry was used to examine HCPT-induced cell cycle block in cultured RLECs. We observed cell morphological changes with light microscope and electron microscope. Apoptosis was detected by terminal deoxyribose transferase-mediated deoxy-uridine triphosphate nick end labeling (TUNEL) assay. The TUNEL assay was performed following the manufacturer's instruction of in situ cell death detection. Expression of Fas and Fas-L was estimated by immunohistochemical staining., Results: The flow cytometric analysis showed that the HCPT induced cell cycle block was at S phase. The ratio of nucleus and cytoplasm was increased. The electron microscopic examinations demonstrated that the HCPT initiated apoptosis with typical morphological features. The apoptosis of RLECs treated with HCPT was observed by TUNEL assay, and the number of the cells with apoptosis was increased with the increase of the HCPT concentration. The HCPT did not affect the gene expression of Fas/FasL., Conclusions: HCPT not only induces mitotic block but also induces apoptosis of RLECs which is not mediated by the Fas/FasL signaling pathway.