Construction of foot-and-mouth disease virus like particles-induced expression vectors and screening of BHK-21 cell pools / 生物工程学报

Transient expression is the major method to express foot-and-mouth disease virus (FMDV) capsid proteins in mammalian cells. To achieve stable expression of FMDV capsid proteins and efficient assembly of virus like particles (VLPs) in cells, the plasmids of piggyBac (PB) transposon-constitutive expression and PB transposon-tetracycline (Tet) inducible expression vectors were constructed. The function of the plasmids was tested by fluorescent proteins. By adding antibiotics, the constitutive cell pools (C-WT, C-L127P) expressing P12A3C (WT/L127P) genes and the inducible cell pools (I-WT, I-L127P) expressing P12A3C (WT/L127P) genes were generated. The genes of green fluorescent protein, 3C protease and reverse tetracycline transactivator (rtTA) were integrated into chromosome, which was confirmed by fluorescence observation and PCR testing. The cell pool I-L127P has a stronger production capacity of capsid proteins and VLPs, which was confirmed by Western blotting and enzyme linked immunosorbent assay (ELISA), respectively. In conclusion, inducing the chromosomal expression of FMDV capsid proteins was firstly reported, which may facilitate the technical process of mammalian production of FMDV VLPs vaccine and the construction of mammalian inducible expression systems for other proteins.

Generation of Monoclonal Antibodies against Non-structural Protein 3AB of Foot-and-Mouth Disease Virus / 中国病毒学·英文版

To identify linear epitopes on the non-structural protein 3AB of foot-and-mouth disease virus (FMDV),BABL/c mice were immunized with the 3AB protein and splenocytes of BALB/c mice were fused with myeloma Sp2/0 cells.Two hybridoma monoclonal antibodies (mAbs) cell lines against the 3AB protein of foot-and-mouth disease virus (FMDV) were obtained,named C6 and E7 respectively.The microneutralization titer was 1∶1024 for mAb C6,and 1∶512 for E7.Both mAbs contain kappa light chains,and were of subclass IgG2b.In order to define the mAbs binding epitopes,the reactivity of these mAbs against FMDV were examined by indirect ELISA.The results showed that both mAbs can react with FMDV,but had no cross-reactivity with Swine Vesicular Disease (SVD) antigens.The titers in abdomen liquor were 1∶5×106 for C6 and 1∶2×106 for E7.In conclusion,the mAbs obtained from this study are specific for the detection of FMDV,can be used for etiological and immunological researches on FMDV,and have potential use in diagnosis and future vaccine designs.

Develope Monoclonal Antibody against Foot-and-mouth Disease Virus A Type / 中国病毒学·英文版

In order to develop an anti-FMDV A Type monoclonal antibo by (mAb),BABL/c mice were immunized with FMDV A type.Monoclonal antibodies (mAbs) 7B11 and 8H4 against Foot-and-mouth disease virus (FMDV) serotype A were produced by fusing SP2/O myeloma cells with splenocyte from the mouse immunized with A/AV88.The microneutralization titer of the mAbs 7B11 and 8H4 were 1024 and 512,respectively.Both mAbs contain kappa light chains,the mAbs were IgG1.In order to define the mAbs binding epitopes,the reactivity of these mAbs against A Type FMDV,were examined using indirect ELISA,the result showed that both mAbs reacted with A Type FMDV.These mAbs may be used for further vaccine studies,diagnostic methods,prophylaxis,etiological and immunological research on FMDV.Characterization of these ncindicated that prepared anti-FMDV A mAbs had no cross-reactivity with Swine Vesicular Disease (SVD) or FMDV O,Asial and C Type antigens.Their titers in abdomen liquor were 1:5×106 and 1:2×106,respectively.7B11 was found to be of subtype IgG1,8H4 was classified as IgG2b subtype.The mAbs prepared in this study,are specific for detection of FMDV serotype A,and is potentially useful for pen-side diagnosis.

A Simple and Rapid Colloidal Gold-based Immunochromatogarpic Strip Test for Detection of FMDV Serotype A / 中国病毒学·英文版

A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test, affinity purified polyclonal antibodies from Guinea pigs which were immunized with sucking-mouse adapted FMD virus (A/AV88(L) strain) were conjugated to colloidal gold beads and used as the capture antibody, and affinity purified polyclonal antibodies from rabbits which were immunized with cell-culture adapted FMD virus (A/CHA/09 strain) were used as detector antibody. On the nitrocellulose membrane of the immunochromatographic strip, the capture antibody was laid on a sample pad, the detector antibody was printed at the test line(T) and goat anti-guinea pigs IgG antibodies were immobilized to the control line(C). The lower detection limit of the test for a FMDV 146S antigen is 11.7ng/ml as determined in serial tests after the strip device was assembled and the assay condition optimization. No cross reactions were found with FMDV serotype C, Swine vesicular disease (SVD), Vesicular stomatiti svirus (VSV) and vesicular exanthema of swine virus (VES) viral antigens with this rapid test. Clinically, the diagnostic sensitivity of this test for FMDV serotypes A was 88.7% which is as same as an indirect-sandwich ELISA. The specificity of this strip test was 98.2% and is comparable to the 98.7% obtained with indirect-sandwich ELISA. This rapid strip test is simple, easy and fast for clinical testing on field sites;no special instruments and skills are required, and the result can be obtained within 15 min. To our knowledge, this is the first rapid immunochromatogarpic assay for serotype A of FMDV.

B Cell Epitopes within VP1 of Type O Foot-and-mouth Disease Virus for Detection of Viral Antibodies / 中国病毒学·英文版

In this study,the coding region of type O FMDV capsid protein VP1 and a series of codon optimized DNA sequences coding for VP1 amino acid residues 141-160(epitopel),tandem repeat 200-213(epitope2(+2))and the combination of two epitopes(epitope1-2)was genetically cloned into the prokaryotic expression vector pPROExHTb and pGEX4T-1,respectively.VP1 and the fused epitopes GST-E1,GST-E2(+2)and GST-E1-2 were successfully solubly expressed in the cytoplasm of Escherichia coli and Western blot analysis demonstrated they retained antigenicity.Indirect VP1-ELISA and epitope ELISAs were subsequently developed to screen a panel of 80 field pig sera using LPB-ELISA as a standard test.For VP1-ELISA and all the epitope ELISAs,there were clear distinctions between the FMDV-positive and the FMDV-negative samples.Cross-reactions with pig sera positive to the viruses of swine vesicular disease virus that produce clinically indistinguishable syndromes in pigs or guinea pig antisera to FMDV strains of type A,C and Asial did not occur.The relative sensitivity and specificity for the GST-E1 ELISA,GST-E2(+2),GST-E1-2 ELISA and VP1-ELISA in comparison with LPB-ELISA were 93.3% and 85.0%,95.0% and 90%,100% and 81.8%,96.6% and 80.9% respectively.This study shows the potential use of the aforementioned epitopes as alternatives to the complex antigens used in current detection for antibody to FMDV structural proteins.

Amplification and Characterization of Bull Semen Infected Naturally with Foot-and-mouth Disease Virus Type Asial by RT-PCR / 中国病毒学·英文版

To investigate the security of semen biologically, 15 bull semen samples were collected (of which 5 exhibited clinical signs of Foot-and-mouth disease) and identified by RT-PCR and virus isolation. The results indicated that the semen of the infected bulls were contaminated by Foot-and-mouth disease virus (FMDV), but FMDV was not detected in semen samples from those bulls not showing clinical signs of Foot-and-mouth disease (FMD). This is the first report of the presence of FMDV in bull semen due to natural infection in China. The analysis of the partial sequence of the VP1 gene showed that the virus strain isolated from semen has 97.9% identity with the virus isolated from vesicular liquid of infected bulls showing typical signs of FMD and belonged to the same gene sub-group.

Comparison of Three ELISA Kits for the Differentiation of Foot-and-mouth Disease Virus-infected from Vaccinated Animals / 中国病毒学

A study was performed to validate 3 FMDV 3ABC-I-ELISA kits developed in China for the differentiation of FMDV infected and vaccinated animals.Sets of sera from naive and vaccinated cattle as well as from cattle that had been infected were tested for antibodies against nonstructural proteins (NSPs) of FMDV by commercial diagnosis kits,Ceditest(R)FMDV-NS (Ceditest(R) kit),UBI(R) FMDV NONSTRUCTURAL PROTEIN ELISA DIRECTION INSERT (UBI(R) kit) and a FMDV 3ABC-I-ELISA kitdeveloped at the Lanzhou Veterinary Research Institute.The test parameters (sensitivity and specificity) of the three kits were determined,and the result obtained from FMD 3ABC-I-ELISA kit was compared with that obtained from two foreign kits.The results indicated that the coincidence rate between the FMDV 3ABC-I-ELISA and Ceditest(R) kits was 98.05%,and the coincidence rate between the FMDV 3ABC-I-ELISA and UBI(R) kits was 94.4%; the sensitivity of both Ceditest(R) and FMDV 3ABC-I-ELISA kit was 100%.However,the sensitivity of the UBI(R) kit was only 81.8%.With sera from naive or vaccinated non-infected animals,the specificity of all tests exceeded 90%.