Qingge Xie

Expression of the major epitope regions of 2C integrated with the 3AB non-structural protein of foot-and-mouth disease virus and its potential for differentiating infected from vaccinated animals.

In recent years, the potential value of the non-structural proteins (NSP) 2C and 3ABC has been well documented for differentiation of animals infected with foot-and-mouth disease virus (FMDV) from vaccinated animals (DIVA). In order to develop a more sensitive approach to detect animals infected naturally in herds of FMDV-vaccinated animals, a 47.6kD fusion protein named 2C3AB was expressed in bacteria which incorporated two major B-cell epitope regions of 2C and the whole 3AB within the NSP of FMDV. The product reacted specifically with sera from animals infected with FMDV, but did not react with sera from non-vaccinated and healthy animals. The performance of 2C3AB was compared further with the 3ABC fusion protein as the antigen in an indirect ELISA format for DIVA. The results showed that the 2C3AB-ELISA had an even stronger signal reaction in the indirect ELISA and showed higher sensitivity than the 3ABC-ELISA for DIVA purposes and for detection of early virus infection in animals. Therefore, it is expected that the recombinant protein 2C3AB could be a good candidate protein with which to develop more sensitive methods for DIVA and for surveillance of herds infected subclinically under conditions of vaccination. This study indicates that the 2C3AB-ELISA can be used to confirm the results of the 3ABC-ELISA to improve the performance of the 3ABC-ELISA DIVA test.

Protection of guinea pigs and swine by a recombinant adenovirus expressing O serotype of foot-and-mouth disease virus whole capsid and 3C protease

Two recombinant adenoviruses were constructed expressing foot-and-mouth disease virus (FMDV) capsid and 3C/3CD proteins in replicative deficient human adenovirus type 5 vector. Guinea pigs vaccinated with 1-3 x 10(8)TCID(50) Ad-P12x3C recombinant adenovirus were completely protected against 10,000GID(50) homologous virulent FMDV challenge 25 days post vaccination (dpv). Ad-P12x3CD vaccinated guinea pigs were only partially protected. Swine were vaccinated once with 1x10(9)TCID(50) Ad-P12x3C hybrid virus and challenged 28 days later. Three of four vaccinated swine were completely protected against 200 pig 50% infectious doses (ID(50)) of homologous FMDV challenge, and vaccinated pigs developed specific cellular and humoral immune responses. The immune effect of Ad-P12x3C in swine further indicated that the recombinant adenovirus was highly efficient in transferring the foreign gene. This approach may thus be a very hopeful tool for developing FMD live virus vector vaccine.

Development of a hamster kidney cell line expressing stably T7 RNA polymerase using retroviral gene transfer technology for efficient rescue of infectious foot-and-mouth disease virus.

Reverse genetics systems, with the ability to manipulate viral genomes at the DNA molecular level, are an important platform for study of the assembly and function of viruses. Genome manipulation, such as gene recombination, mosaicism, and mutation may interfere with replication, assembly and release of viruses. An efficient, convenient and economical method of virus rescue is undoubtedly required for increasing the efficiency of rescuing recombinant viruses. To develop an efficient, helper virus-free viral recovery system (reverse genetics), a retroviral gene transfer technology was used to establish a stable BHK-21 cell line (designated as BHKT7) which expressed constitutively bacteriophage T7 RNA polymerase (T7 RNAP). An improved method for rescue of infectious foot-and-mouth disease virus (FMDV) was then developed. FMDV full-length cDNA under control of a T7 promotor, was transfected into BHKT7 of differing passages. FMDV virus was rescued efficiently from the BHKT7 cells, the passage number not having an effect on the efficiency of recovery. As a result, the cell line was stable even after multiple passages, expressing sufficient T7 RNAP to support ex vivo transcription and efficient rescue. The reverse genetics system described below is efficient, stable, and convenient. The system could provide not only the basis of gene function research into FMDV, but could also be used for reverse genetics research into other positive-strand RNA viruses, without the need for helper viruses.

Complete nucleotide sequence of a Chinese serotype Asia1 vaccine strain of foot-and-mouth disease virus.

The full-length nucleotide sequence of the Chinese vaccine strain Asia1/YNBS/58 of foot-and-mouth disease virus (FMDV) was determined. The results showed that the complete genome of YNBS/58 is 8,164 nucleotides (nt) in length including a 1,061-nt 5′ untranslated region (UTR), a 6,990-nt open reading frame (ORF), and a 113-nt 3′ UTR. Genome sequences of Asia1/YNBS/58 and other known FMDV strains were compared. The homology analysis indicated that non-structural proteins are more conserved than structural proteins in FMDV and that the 5′ UTR is more conserved than the 3′ UTR. Phylogenetic analysis revealed that Asia1/YNBS/58 is clustered in the Asia1 serotype and is linked to three other isolates, Asia1/IND/63/72, Asia1/3kimron/63, and Asia1/2ISRL/63, suggesting that they have a close genetic relationship. The VP1-, VP2-, and VP3-based phylogenetic trees divided into distinct clusters according to the different serotypes, while other gene-based phylogenetic trees exhibited some degree of intercrossing among serotypes. According to the nucleotide similarities between Asia1/YNBS/58 and two recent Chinese Asia1 isolates, Asia1/HKN/05, and Asia1/JS/05, each forms a distinct genotype. This study is the first description of the full-length genomic sequence of FMDV Chinese serotype Asia1.

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 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 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.

Integrin Activation and Viral Infection

Integrins are members of a ubiquitous membrane receptor family which includes 18 different α subunits and 8 β subunits forming more than 20 α/β heterodimers. Integrins play key functions in vascular endothelial cell and tumour cell adhesion, lymphocyte trafficking, tumor growth and viral infection. Current understanding of the molecular basis of integrins as viral receptors has been achieved through many decades of study into the biology of transmembrane glycoproteins and their interactions with several viruses. This review provides a summary of the current knowledge on the molecular bases of interactions between viruses and integrins, which are of potential practical significance. Inhibition of virus-integrin interactions at the points of virus attachment or entry will provide a novel approach for the therapeutic treatment of viral diseases.

Simple Method of Monoclonal Antibody Production Against Mammalian Cellular Prion Protein

Monoclonal antibodies (MAbs) against prion protein (PrP) are powerful tools for diagnosis and research in transmissible spongiform encephalopathies. Ten MAbs to recombinant/native cellular PrP (PrPc) in mammals were prepared with a simple method and identified in detail. Normal BALB/c mice were immunized with the recombinant bovine mature PrP (rbomPrP) and PrP27-30 (rboPrP27-30) expressed in Escherichia coli. The immunized splenocytes were fused with SP2/0 mouse myeloma cells, and positive hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA). The characterizations of these MAbs, such as Ig, Ig subclass, titer, affinity index, specificity, epitopes recognized, and binding to recombinant/native PrPc of cattle, sheep, or human beings, were evaluated by Western blotting and indirect or sandwich ELISA. Ten MAbs could be divided into five groups depending on the results of indirect ELISA additivity test and their reaction to E. coli-expressed truncated-PrPs. Isotyping of the MAbs revealed that they belong to IgG1, IgG2a, and IgG2b subclass. Their indirect ELISA titers were between 10(3) and 10(6). Affinity constants were between 10(9) and 10(12) M(-1). Ten MAbs specifically reacted with the rbomPrP, without binding to prion-like protein Doppel and the lysates of E. coli. These MAbs could also respond to the recombinant mature PrP (rmPrP) of sheep and human beings. Also of interest was the ability of the MAbs to bind with dimer of rmPrP and PrP extracted from the brain tissue of cattle or sheep. We conclude that anti-PrP MAbs successfully prepared with a simple method could potentially be useful in mammalian prion research.

Expression of major antigen domains of E2 gene of CSFV and analysis of its immunological activity

E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection.

Construction of Recombinant Retroviral Vector Carrying Lab Gene of Foot-and-mouth Disease Virus and Its Expression in Bovine Kidney (MDBK) Cells

In this study, foot-and-mouth disease virus (FMDV) strain OA/58 RNAs were used as templates for RT-PCR. By the molecular cloning, the Lab gene encoding leader protease called Lpro were cloned in retroviral vector pBPSTR1 to obtain reconstruction retroviral vector termed pBPSTR1-Lab. At different concentrations of puromycin and tetracycline respectively in the cell culture mediums, the growth of bovine kidney cells (MDBK) showed that the optimal puromycin resistant selection concentration was 3 microg/mL and tetracycline regulatory concentration was 1 microg/mL. Pseudotyped retroviral virus particles were produced by transiently co-tansfecting GP2-293 cells with a retroviral vector DNA and VSV-G plasmid. Then MDBK cells were infected by pseudotyped retroviral virus and were continually seeded in the medium at the optimal tetracycline regulatory concentration and puromycin selection concentration for 12 days to obtain puromycin resistant colonies whose genomes contained the Lab gene. After tetracycline removal, synthesis of Lpro induced severe morphological changes in the puromycin resistant MDBK cells. PCR and Western blotting proved that a stable MDBK cell line inducibly expressing the Lab gene under the control of tetracycline was obtained. The experiment might provide a basis for studying that Lpro of FMDV plays an important role in MDBK cell pathogenesis.