[Protective immune response of guinea pigs against challenge with foot and mouth disease virus by immunization with foliar extracts from transgenic tomato plants expressing the FMDV structural protein VP1].

The plant constitutive expression vector pBin438/VP1 for VP1 gene of foot-and-mouth disease virus strain O/ China/99 was constructed. Mediated with Agrobacterium tumefaciens GV3101 harboring pBin438/VP1, VP1 gene was transferred into cotyledons of tomato. After selected by Kanamysin, sixty resistant lines were obtained. The integration and transcription of the VP1 gene in transformed plants was detected by PCR and RT-PCR. After being detected by sandwich-ELISA assays, about 40% transformed plants confirmed to express the recombinant protein. The leave extracts of two positive lines were respectively emulsified in Freund's adjuvant and guinea pigs were intramuscular inoculation at days 0, 15 and 30d. According to the sera antibody levels and the protection of the vaccinated guinea pigs against challenge with 100ID50 FMDV, probed into the immunogenicity of the target protein expressed in transgenic plants. Experimental results showed that the plant expression vector was successfully constructed. PCR and RT-PCR analyses confirmed VP1 gene was transformed into tomato plants and got expression at the transcription levels. The expressed VP1 protein of FMDV, which was identified by ELISA and Western blot, can be specifically recognized by polyclonal antibodies against FMDV. Indirect-ELISA antibody titers reached 1:64 twenty-one days after the third inoculation. In the challenge test, the protection against FMDV challenge in two groups was 80% and 40% respectively. The immunization test in guinea pigs indicated that the expression product of transgenic tomato plants had immunogenicity and could effectively induce the specific antibodies against FMDV.

[Prokaryotic expression, protein purification and reactivity analysis of the VP1 and its C terminus of a South African isolate of FMDV serotype SAT2].

AIM: To induce the expression of structure protein VP1 and its C terminus of foot-and-mouth disease virus (FMDV) serotype SAT2 in E.coli and analyze their reactivities with FMDV positive antiserum. METHODS: The plasmid pGEM-SAT2P1 carrying the VP1 coding region of FMDV serotype SAT2 isolated from South African was used as template for RT-PCR to get the coding fragment of VP1 and its C terminus. The fragments were then cloned into expression vector pET-30a(+) to get recombinant plasmids pET-SAT2VP1 and pET-SAT2VP1C. The recombinant plasmids were transformed into E.coli BL21(DE3)pLysS and induced by IPTG to express VP1 and its C terminus protein. The expressed VP1 and its C terminus were then purified by Ni-NTA His Bind Resin affinity chromatography and analyzed by Western blot. New Zealand rabbits were immunized to prepare polyclonal antibodies against VP1 and VP1C. The antisera were obtained and polyclonal antibody was characterized by ELISA. RESULTS: SDS-PAGE demonstrated that VP1and its C terminus expressed in the E.coli transformants had a molecular weight of 33000 and 19000 and contained in the inclusion body. Purified VP1 and its C terminus was obtained by Ni-NTA His Bind Resin affinity chromatography and a single clear band appeared in the SDS-PAGE gel. Western blot analysis showed that the purified VP1 and VP1 C terminus could react with bovine antiserum against the same serotype FMDV without cross-reactivity with the negative bovine serum. CONCLUSION: Rabbit polyclonal antibodies against VP1 and VP1C were successfully prepared, the titers of which were above 1:12800 and had obvious specificity.

B cell epitopes within VP1 of type O foot-and-mouth disease virus for detection of viral antibodies.

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 (epitope1), tandem repeat 200-213 (epitope2 (+2)) and the combination of two epitopes (epitope1-2) was genetically cloned into the prokaryotic expression vector pP(RO)ExHTb 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 Asia1 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.

The efficacy of FMD vaccine reduced non-structural proteins with a mAb against 3B protein.

A monoclonal antibody, 3BIgG, against the prokaryotically expressed foot-and-mouth disease virus (FMDV) non-structural protein (NSP) 3B was obtained. The 3BIgG-sepharose conjugant (3BmAb-6BFF) was prepared by adding the purified 3BIgG into epoxy-activated sepharose 6BFF, incubating with the inactivated FMDV, and then removing the sepharose by centrifugation. The vaccine was made from the supernatant emulsified with oil-adjuvant ISA206. Ten guinea pigs, 26 pigs and six cattle were vaccinated, and a vaccination control group was included without treatment with 3BmAb-6BFF. After 28 days, 9/10 pigs challenged with FMDV were protected, this result was the same as the control group, indicating that the vaccine potency was not reduced after treatment with 3BmAb-6BFF. The other animals were vaccinated weekly for nine weeks, and serum samples were collected to detect 3ABC-antibody titers. The results showed that 3ABC-antibody production was delayed and the positive antibody rates were lower when vaccination was carried out using vaccines treated with 3BmAb-6BFF compared with untreated vaccines. The findings of this study suggest that it is possible to reduce NSPs using a mAb-sepharose conjugant in FMD vaccines without reducing their efficacy.

[Genome sequencing and analysis of foot-and-mouth disease virus Asia1/YNBS/58 strain].

The full-length genomic sequence of foot-and-mouth disease virus (FMDV) Asia1/YNBS/58 strain was determined by RT-PCR and compared with other 17 reference strains. The results showed that the complete genome of Asia1/YNBS/58 was 8164nt long including a 1061-nt 5' untranslated region (UTR), a 6990-nt open reading frame (ORF), and a 113-nt 3'UTR. The homology analysis indicated that the UTR regions and non-structural proteins were more conserved than the structural proteins in FMDV. VP1 exhibited the lowest conservation and VP4 was exceptionally conserved. The VP1-, VP2-, and VP3-based phylogenetic trees were divided into distinct clusters according to different serotypes, while the other gene-based phylogenetic trees exhibited some degree of intercross among serotypes. This study is the first description of the full-length genomic sequence of FMDV Chinese serotype Asia1.

[Molecular cloning and characteristics of cDNA encoding pig beta6 subunit for FMDV receptor].

In order to study the roles of integrin beta6 in Foot-and-Mouth Disease Virus infection, pig integrin beta6 was firstly molecularly cloned from RNA of the tongue and lung of recovered pig infected experimentally with foot-and-mouth-disease virus (FMDV), and was compared with the beta6 gene of other animals available in GenBank at nucleotide and amino acid leves. GeneBank association number of the beta6 gene is EF432729. Pig integrin beta6 gene (2367bp) encodes a polypeptide of 788 amino acids consisting of 9 potential N-linked glycosylation sites, 3 Glycosaminoglycan attachment sites, a cGMP-dependent protein kinase phosphorylation site, 10 Protein kinase C phosphorylation sites, 2 EGF-like domains and 2 cysteine-rich regions. Pig integrin beta6 subunit has a 26-residue putative signal peptide, a 681-residue ectodomain, a 29-residue transmembrane domain, and a 52-residue cytoplasmic domain. 11 mutant nucleotides were found in beta6 gene coding region and 9 amino acids were changed. The nucleotide sequence similarity of integrin beta6 gene between rheses monkey, mouse, Norway rat, dog, guinea pig, human, bovine, sheep is 79.5%, 84.9%, 85.4%, 85.2%, 88.7%, 90.1%, 91.9% and 91.9%, and the amino acid sequence similarity is 93.5%, 88.2%, 88.5%, 88.3%, 91.0%, 92.8%, 93.3% and 93.4% respectively. This study will lay a foundation for understanding the interactions of FMDV with receptors.

[Establishment of indirect ELISA diagnose based on the VP1 structural protein of foot-and-mouth disease virus (FMDV) in pigs].

The complete gene encoding the structural protein of FMDV(VP1) was subcloned into expression vector pPROex-HT, resulting in the fusion expression plasmid pPROexHT-VP1. After transformed into E. coli BL21(DE3) and induced by IPTG, the fusion protein was expressed in high level. Western blot was performed to confirm that the expressed fusion protein could specifically react with antiserum against FMDV. Based on the fusion protein further purified, a novel indirect ELISA (VP1-ELISA) was developed to detect FMDV antibody in pigs. Comparison between VPl-ELISA and the government standard kit (liquid phase block ELISA) showed the two methods had 96.25 percent agreement by detecting 80 serum samples, indicating that the indirect VP1-ELISA was specific and sensitive.

[Cloning and sequence analysis of cDNA encoding porcine alphav subunit for FMDV receptor].

Receptors play a crucial role in determining the pathogenesis and tissue tropism of virus. Foot-and-mouth disease virus (FMDV) has been showed to use four integrins, alphavbeta1, alphavbeta3, alphavbeta6 and alphavbeta8 as receptors to initiate infection. In this study, the porcine integrin alphav gene was cloned by RT-PCR from the lung tissue of healed pig infected experimently with FMDV, and compared its nucleotide and deduced amino acid sequence with the av gene of other animals. The 3141bp cDNA of bovine integrin alphav encodes a polypeptide of 1046 amino acids consisting of a 30-residue putative signal peptide, a 955-residue ectodomain, a 29-residue transmembrane domain, and a 32-residue cytoplasmic domain. The ectodomain contains 11 potential N-linked glycosylation sites (NXT/NXS), 2 calcium binding domains (DX[D/N] XDGXXD) and 18 cysteine residues. The nucleotide sequence similarities of integrin alphav between pig and cattle, human, rheses monkey, house mouse, chicken, dog are 93.3%, 91.5%, 91.4%, 85.6%, 73.2% and 89.9% respectively; and the amino acid sequence similarities are 96.3%, 94.6%, 94.1%, 90.8%, 81.6% and 93.8%, respectively. The alphav gene of cattle and pig exhibited the highest sequence homology. It is possible that host tropism of FMDV may related to divergence in receptors among different species.

The effect of bovine IFN-alpha on the immune response in guinea pigs vaccinated with DNA vaccine of foot-and-mouth disease virus.

In this study, we constructed recombinant plasmid pcDNA3.1/P12X3C3D including P1, 2A, 3C, 3D and part of 2B gene of FMDV and pcDNA3.1/IFN containing the gene encoding bovine IFN-alpha. We inoculated the DNA vaccine pcDNA3.1/P12X3C3D with or without pcDNA3.1/IFN to evaluate the efficiency of this DNA vaccine and the immunogenicity of DNA vaccine enhanced by the co-delivery with pcDNA3.1/IFN. After two times of vaccination with DNA vaccine, all of guinea pigs were challenged with 103 ID50 FMDV type O. Anti-FMDV antibody levels were detected by ELISA and T lymphocyte proliferation response was tested by MTT assay. The result shows that guinea pigs inoculated by pcDNA3.1/P12X3C3D alone or with pcDNA3.1/IFN generated specific antibodies and induced an FMDV-specific T lymphocyte proliferation response. FMDV challenge tests showed that one in four guinea pigs immunized by pcDNA3.1/P12X3C3D with pcDNA3.1/IFN was protected from the FMDV serotype O infection. This result indicated that the efficiency of the DNA vaccine was enhanced by co-delivery with pcDNA3.1/IFN. However, the protection rate was considerably lower than that immunized with conventional FMD vaccine.

[Expression of recombinant plasmid pcDNA3.1/P12X3C with multi-genes of foot-and-mouth disease virus in BHK-21 cells].

In order to obtain the gene P12X3C of Foot-and-Mouth Disease Virus (FMDV) that includes full length P1, 2A, 3C and a part of 2B, the site mutation strategy was used. After being digested by Kpn I and Xba I respectively, the gene P12X3C was cloned into the pcDNA3.1 (+) expression vector. The recombinant plasmid was checked by restriction enzyme analysis and nucleic acid sequencing, and then named pcDNA3.1/P12X3C. Further, BHK-21 cells was transfected with pcDNA3.1/P12X3C by using lipoid. The proteins of Foot-and-Mouth Disease Virus, which were expressed in BHK-21 cells, were confirmed by sandwich-ELISA and fluoroscopy. The result shows the gene P12X3C is cloned into eukaryotic expression plasmid, and the recombinant eukaryotic expression plasmid pcDNA3.1/P12X3C could express proteins of Foot-and-Mouth Disease Virus in BHK-21 cells, which have immunocompetence. This study demonstrates that delivery of a recombinant eukaryotic expression plasmid containing P12X3C coding regions results in the assembly of FMDV capsid structures, which will offer experimental base to DNA vaccine of FMDV.