Subclinical bluetongue virus infection in domestic ruminants in Taiwan.

Bluetongue is an arthropod-borne viral disease affecting domestic and wild ruminants. Taiwan, with the Tropic of Cancer crossing through it, was considered free of bluetongue virus (BTV) before 2001. The goals of this study are to identify the serotype and phylogeny of Taiwan BTV isolates and to understand the serological status and chronology of BTV infection. Analysis of the S10 gene segment revealed that Taiwan BTV isolates are closely related to Chinese strains. Seropositive results were found in 32.7% of the cattle and 8.2% of the goats by head, and 90.7% of the cattle herds and 28.9% of the goat flocks. Anti-BTV antibodies have existed in goat sera since 1989 and in bovine sera since 1993, and over the years, the seropositive rates in rapidly urbanized districts have decreased, most likely due to the loss of vector habitats. Seropositive rates for sheep were variable, due to a small sample size and a small sheep population. Thus far, all natural BTV infections have been subclinical, consistent with experimental sheep inoculation, revealing that the Taiwan isolate is of low virulence.

Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes.

BACKGROUND: Plant viruses can be employed as versatile vectors for the production of vaccines by expressing immunogenic epitopes on the surface of chimeric viral particles. Although several viruses, including tobacco mosaic virus, potato virus X and cowpea mosaic virus, have been developed as vectors, we aimed to develop a new viral vaccine delivery system, a bamboo mosaic virus (BaMV), that would carry larger transgene loads, and generate better immunity in the target animals with fewer adverse environmental effects. METHODS: We engineered the BaMV as a vaccine vector expressing the antigenic epitope(s) of the capsid protein VP1 of foot-and-mouth disease virus (FMDV). The recombinant BaMV plasmid (pBVP1) was constructed by replacing DNA encoding the 35 N-terminal amino acid residues of the BaMV coat protein with that encoding 37 amino acid residues (T128-N164) of FMDV VP1. RESULTS: The pBVP1 was able to infect host plants and to generate a chimeric virion BVP1 expressing VP1 epitopes in its coat protein. Inoculation of swine with BVP1 virions resulted in the production of anti-FMDV neutralizing antibodies. Real-time PCR analysis of peripheral blood mononuclear cells from the BVP1-immunized swine revealed that they produced VP1-specific IFN-gamma. Furthermore, all BVP1-immunized swine were protected against FMDV challenge. CONCLUSION: Chimeric BaMV virions that express partial sequence of FMDV VP1 can effectively induce not only humoral and cell-mediated immune responses but also full protection against FMDV in target animals. This BaMV-based vector technology may be applied to other vaccines that require correct expression of antigens on chimeric viral particles.

Comparative studies of the capsid precursor polypeptide P1 and the capsid protein VP1 cDNA vectors for DNA vaccination against foot-and-mouth disease virus.

BACKGROUND: Foot-and-mouth disease virus (FMDV) causes a severe livestock disease, and the virus is an interesting target for virology and vaccine studies. MATERIALS AND METHODS: Here we evaluated comparatively three different viral antigen-encoding DNA sequences, delivered via two physical means (i.e., gene gun delivery into skin and electroporation delivery into muscle), for naked DNA-mediated vaccination in a mouse system. RESULTS: Both methods gave similar results, demonstrating commonality of the observed DNA vaccine effects. Immunization with a cDNA vector expressing the major viral antigen (VP1) alone routinely failed to induce the production of anti-VP1 or neutralizing antibodies in test mice. As a second approach, the plasmid L-VP1 that produces a transgenic membrane-anchored VP1 protein elicited a strong antibody response, but all test mice failed in the FMDV challenge experiment. In contrast, for mice immunized with the viral capsid precursor protein (P1) cDNA expression vector, both neutralizing antibodies and 80-100% protection in test mice were detected. CONCLUSIONS: This strategy of using the whole capsid precursor protein P1 cDNA for vaccination, intentionally without the use of virus-specific protease or other encoding genes for safety reasons, may thus be employed as a relevant experimental system for induction or upgrading of effective neutralizing antibody response, and as a convenient surrogate test system for DNA vaccination studies of FMDV and presumably other viral diseases.

Induction of immunity in swine by purified recombinant VP1 of foot-and-mouth disease virus.

VP1, a capsid protein of foot-and-mouth disease virus (FMDV), contains neutralizing epitopes of the virus. Due to its poor water solubility, recombinant Escherichia coli derived VP1 (rVP1) has previously been used mainly in a denatured form and is not well characterized. Here, using SDS to assist protein refolding and then removing SDS with a detergent removing column, we have successfully purified rVP1 in two aqueous-soluble forms, i.e. monomer and dimer. Studies showed that dimerization occurs by an inter-molecular disulfide bond between two cysteine residues at position 187 of each monomer. Heat treatment revealed that rVP1 dimer exhibited a more thermal-stable conformation than the monomeric form. Both monomeric and dimeric rVP1 reacted with anti-FMDV antibodies. Immunization studies demonstrated that vaccination of swine with either forms of rVP1 was effective in generating immune responses and protecting them from viral challenge.

Effective synthetic peptide vaccine for foot-and-mouth disease in swine.

We have designed a peptide-based vaccine for foot-and-mouth disease (FMD) effective in swine. The peptide immunogen has a G-H loop domain from the VP1 capsid protein of foot-and-mouth disease virus (FMDV) and a novel promiscuous T helper (Th) site for broad immunogenicity in multiple species. The G-H loop VP1 site was optimised for cross-reactivity to FMDV by the inclusion into the peptide of cyclic constraint and adjoining sequences. The incorporation of consensus residues into the hypervariable positions of the VP1 site provided for broad immunogenicity. The vaccine protected 20 out of 21 immunised pigs from infectious challenge by FMDV O1 Taiwan using peptide doses as low as 12.5 microg, and a mild adjuvant that caused no lesions. A safe chemically-defined product would have considerable advantages for vaccination against FMD.

Anti-3AB antibodies in the Chinese yellow cattle infected by the O/Taiwan/99 foot-and-mouth disease virus.

The O/Taiwan/99 foot-and-mouth disease virus (FMDV), a South Asian topotype of serotype O, was introduced into Taiwan in 1999. The Chinese yellow cattle infected by the virus did not develop clinical lesions under experimental and field conditions. A blocking enzyme-linked immunosorbent assay (ELISA) kit with the 3AB antigen, a polypeptide of FMDV non-structural (NS) proteins, was used to evaluate the development and duration of anti-3AB antibodies, proving active viral replication, in the Chinese yellow cattle. The specificity of the assay was 99%, as was established with negative sera from regularly vaccinated and from naïve cattle. The sensitivity tested with sera from naturally infected animals was approximately 64% and it was lower than that obtained by serum neutralization (SN) test. Under experimental infection, the Chinese yellow cattle developed lower anti-3AB antibodies than that developed in other species. Duration of anti-3AB antibodies was traced in two herds of naturally infected animals, indicating that anti-3AB antibodies persisted for approximately 6 months after outbreaks. On the basis of this study, we propose that the Chinese yellow cattle may have natural resistance, which limits viral replication and reduces the development of anti-3AB antibodies.