Capsid Protein Precursor Research Articles

Genome organization and translation products of Providence virus: insight into a unique tetravirus

Providence virus (PrV) is a member of the family Tetraviridae, a family of small, positive-sense, ssRNA viruses that exclusively infect lepidopteran insects. PrV is the only known tetravirus that replicates in tissue culture. We have analysed the genome and characterized the viral translation products, showing that PrV has a monopartite genome encoding three ORFs: (i) p130, unique to PrV and of unknown function; (ii) p104, which contains a read-through stop signal, producing an N-terminal product of 40 kDa (p40) and (iii) the capsid protein precursor (p81). There are three 2A-like processing sequences: one at the N terminus of p130 (PrV-2A₁) and two more (PrV-2A₂ and PrV-2A₃) at the N terminus of p81. Metabolic radiolabelling identified viral translation products corresponding to all three ORFs in persistently infected cells and showed that the read-through stop in p104 and PrV-2A₃ in p81 are functional in vivo and these results were confirmed by in vitro translation experiments. The RNA-dependent RNA polymerase domain of the PrV replicase is phylogenetically most closely related to members of the families Tombusviridae and Umbraviridae rather than to members of the family Tetraviridae. The unique genome organization, translational control systems and phylogenetic relationship with the replicases of (+ve) plant viruses lead us to propose that PrV represents a novel family of small insect RNA viruses, distinct from current members of the family Tetraviridae.

Expression and immunological analysis of capsid protein precursor of swine vesicular disease virus HK/70

VP1, a capsid protein of swine vesicular disease virus, was cloned from the SVDV HK/70 strain and inserted into retroviral vector pBABE puro, and expressed in PK15 cells by an retroviral expression system. The ability of the VP1 protein to induce an immune response was then evaluated in guinea pigs. Western blot and ELISA results indicated that the VP1 protein can be recognized by SVDV positive serum, Furthermore, anti-SVDV specific antibodies and lymphocyte proliferation were elicited and increased by VP1 protein after vaccination. These results encourage further work towards the development of a vaccine against SVDV infection.

Effect of foot-and-mouth disease virus capsid precursor protein and 3C protease expression on bovine herpesvirus 1 replication

Several reports have previously shown that expression of the foot-and-mouth disease virus (FMDV) capsid precursor protein encoding region P1-2A together with the 3C protease (P1-2A/3C) results in correct processing of the capsid precursor into VP0, VP1 and VP3 and formation of FMDV capsid structures that are able to induce a protective immune response against FMDV challenge after immunization using naked DNA constructs or recombinant viruses. To elucidate whether bovine herpesvirus 1 (BHV-1) might also be suitable as a viral vector for empty capsid generation, we aimed to integrate a P1-2A/3C expression cassette into the BHV-1 genome, which, however, failed repeatedly. In contrast, BHV-1 recombinants that expressed an inactive 3C protease or the P1-2A polyprotein alone could be easily generated, although the recombinant that expressed P1-2A exhibited a defect in direct cell-cell spread and release of infectious particles. These results suggested that expression of the original, active FMDV 3C protease is not compatible with BHV-1 replication. This conclusion is supported by the isolation of recombinant BHV-1/3C*, which contained mutations within the 3C ORF (3C* ORF)--probably introduced spontaneously during generation of BHV-1/3C*--instead of the authentic 3C ORF contained in the transfer plasmids. Within the 3C* ORF, the codons for glycine 38 and phenylalanine 48 were both substituted by codons for serine. The resulting 3C* protease exhibits a highly reduced activity for proteolytic processing of the P1-2A polyprotein and thus might be a good candidate for the generation of live attenuated FMDV variants.

Foot-and-Mouth Disease Virus Assembly: Processing of Recombinant Capsid Precursor by Exogenous Protease Induces Self-Assembly of Pentamers In Vitro in a Myristoylation-Dependent Manner

The assembly of foot-and-mouth disease virus (FMDV) particles is poorly understood. In addition, there are important differences in the antigenic and receptor binding properties of virus assembly and dissociation intermediates, and these also remain unexplained. We have established an experimental model in which the antigenicity, receptor binding characteristics, and in vitro assembly of capsid precursor can be studied entirely from purified components. Recombinant capsid precursor protein (P1 region) was expressed in Escherichia coli as myristoylated or unmyristoylated protein. The protein sedimented in sucrose gradients at 5S and reacted with monoclonal antibodies which recognize conformational or linear antigen determinants on the virion surface. In addition, it bound the integrin alpha(v)beta(6), a cellular receptor for FMDV, indicating that unprocessed recombinant capsid precursor is both structurally and antigenically similar to native virus capsid. These characteristics were not dependent on the presence of 2A at the C terminus but were altered by N-terminal myristoylation and in mutant precursors which lacked VP4. Proteolytic processing of myristoylated precursor by recombinant FMDV 3C(pro) in vitro induced a shift in sedimentation from 5S to 12S, indicating assembly into pentameric capsid subunits. Nonmyristoylated precursor still assembled into higher-order structures after processing with 3C(pro), but these particles sedimented in sucrose gradients at approximately 17S. In contrast, mutant precursors lacking VP4 were antigenically distinct, were unable to form pentamers, and had reduced capacity for binding integrin receptor. These studies demonstrate the utility of recombinant capsid precursor protein for investigating the initial stages of assembly of FMDV and other picornaviruses.

Construction and immune response characterization of a recombinant pseudorabies virus co-expressing capsid precursor protein (P1) and a multiepitope peptide of foot-and-mouth disease virus in swine

Foot-and-mouth disease (FMD) is the most contagious and devastating disease of livestock. Our previous studies demonstrated that TK(-)/gG(-)/P1(+), a recombinant expressing the FMDV capsid precursor protein (P1) based on attenuated pseudorabies virus (PRV) TK(-)/gG(-), could be used as a recombinant vaccine to protect pigs against both pseudorabies and FMD. However, because the P1 expression cassette is inserted into the gG locus of the genome of PRV TK(-)/gG(-), this bivalent vaccine cannot be used in conjunction with the PRV gE-ELISA, an extensively used discriminatory serological test in eradication programs for pseudorabies, which limits the clinical use of this bivalent vaccine. To circumvent this shortcoming, in this study, an expression cassette containing synthetic multiepitope gene "FHG" consisting of six potential B cell epitopes and two potential T cell epitopes of FMDV, under the control of CMV promoter, was further inserted into the gE/gI locus of genome of TK(-)/gG(-)/P1(+), resulting in a new recombinant FHG/P1/PRV. The immunogenicity of FHG/P1/PRV was evaluated and compared with TK(-)/gG(-)/P1(+) in piglets. Our results clearly showed that FHG/P1/PRV performed better than or comparable with TK(-)/gG(-)/P1(+), as demonstrated by comparable PRV-specific neutralizing antibodies, enhanced FMDV-specific neutralizing antibodies, and cellular immune responses. More importantly, no gE- and gG-specific antibodies could be detected in pigs immunized with FHG/P1/PRV. These data indicate that FHG/P1/PRV is a promising bivalent vaccine candidate with more extensive potential application than TK(-)/gG(-)/P1(+) against both pseudorabies and FMD.

The mechanism of an exceptional case of reinitiation after translation of a long ORF reveals why such events do not generally occur in mammalian mRNA translation.

The subgenomic mRNA of feline caliciviruses is bicistronic with the two cistrons overlapping by four nucleotides, ..AUGA. The upstream cistron encodes a 75-kDa major capsid protein precursor (pre-VP1), and the downstream cistron a 10-kDa minor capsid protein. The kinetics of translation in reticulocyte lysates show that the downstream cistron is translated by a termination-reinitiation process, which is unusual in not requiring eIF4G or the eIF4F complex. Reinitiation requires the 3'-terminal 87 nucleotides (nt) of the pre-VP1 ORF, but no other viral sequences. The reinitiation site is selected by virtue of its proximity to this 87-nt element, and not its proximity to the pre-VP1 ORF stop codon, although this must be located not more than approximately 30 nt downstream from the restart codon. This 87-nt element was shown to bind 40S ribosomal subunits and initiation factor eIF3, and addition of supplementary eIF3 enhanced reinitiation efficiency. Mutants defective in reinitiation showed reduced affinity for eIF3 or defective 40S subunit binding (or both). These results suggest a mechanism in which some of the eIF3/40S complexes formed during disassembly of post-termination ribosomes bind to this 87-nt element in a position appropriate for reinitiation following acquisition of an eIF2/GTP/Met-tRNA i ternary complex.

Genome and polypeptides characterization of Tellina virus 1 reveals a fifth genetic cluster in the Birnaviridae family

We characterized tellina virus 1 (TV-1), a birnavirus isolated from the marine bivalve mollusk Tellina tenuis. Genome sequence analysis established that TV-1 is representative of a viral cluster distant from other birnaviruses. The maturation process of the polyprotein encoded by the genomic segment A was delineated with the identification of the N-termini of the viral protease VP4 and the ribonucleoprotein VP3, and the characterization of peptides deriving from the processing of pVP2, the VP2 capsid protein precursor. One of these peptides was shown to possess a membrane-disrupting domain. Like the blotched snakehead virus, the polyprotein exhibits a non-structural polypeptide (named [X]) located between pVP2 and VP4. Mutagenesis analysis allowed the identification in VP4 of a catalytic Ser-Lys dyad that does not possess the common Gly-X-Ser signature of the serine hydrolases. The genomic segment B encodes the viral RNA-dependent RNA-polymerase VP1 with the unique sequence motif arrangement identified in other birnavirus VP1s.

Comparison of immune responses to different foot-and-mouth disease genetically engineered vaccines in guinea pigs

The P12A3C gene from FMDV (serotype O) encoding the capsid precursor protein, and the highly immunogenic gene FHG, which encodes multiple epitopes of FMDV capsid proteins, were inserted into eukaryotic expression vectors to compare different candidate genetically engineered vaccines for foot-and-mouth disease (FMD). A modified live pseudorabies virus (MLPRV) was also used to deliver P12A3C. Guinea pigs were inoculated intramuscularly with the candidate vaccines to compare the ability to elicit immunity of the DNA vector and a live viral vector. An indirect enzyme-linked immunosorbent assay (iELISA), virus-neutralization test and lymphoproliferation assay were used to detect antibody and cellular responses. The group immunized with P12A3C delivered by MLPRV produced significantly greater antibody and cellular responses indicating that MLPRV has a greater ability to mediate exogenous gene delivery than the plasmid DNA vector. Comparison of the immune responses induced by P12A3C and FHG, which were both mediated by DNA plasmids, showed that FHG and P12A3C elicited similar cellular responses, while P12A3C induced higher antibody levels, suggesting that P12A3C is a more powerful immunogen than FHG. In challenge experiments, guinea pigs vaccinated with P12A3C delivered by MLPRV were protected fully from FMDV challenge, whereas guinea pigs vaccinated with P12A3C or FHG delivered by DNA plasmid were only protected partially. This study provides a basis for future construction of a genetically engineered vaccine for FMDV.

Mutation in the Glycosylated Gag Protein of Murine Leukemia Virus Results in Reduced In Vivo Infectivity and a Novel Defect in Viral Budding or Release

All gammaretroviruses, including murine leukemia viruses (MuLVs), feline leukemia viruses, and gibbon-ape leukemia virus, encode an alternate, glycosylated form of Gag polyprotein (glyco-Gag or gPr80gag) in addition to the polyprotein precursor of the viral capsid proteins (Pr65gag). gPr80gag is translated from an upstream in-frame CUG initiation codon, in contrast to the AUG codon used for Pr65gag. The role of glyco-Gag in MuLV replication has been unclear, since gPr80gag-negative Moloney MuLV (M-MuLV) mutants are replication competent in vitro and pathogenic in vivo. However, reversion to the wild type is frequently observed in vivo. In these experiments, in vivo inoculation of a gPr80gag mutant, Ab-X-M-MuLV, showed substantially lower (2 log) initial infectivity in newborn NIH Swiss mice than that of wild-type virus, and revertants to the wild type could be detected by PCR cloning and DNA sequencing as early as 15 days postinfection. Atomic force microscopy of Ab-X-M-MuLV-infected producer cells or of the PA317 amphotropic MuLV-based vector packaging line (also gPr80gag negative) revealed the presence of tube-like viral structures on the cell surface. In contrast, wild-type virus-infected cells showed the typical spherical, 145-nm particles observed previously. Expression of gPr80gag in PA317 cells converted the tube-like structures to typical spherical particles. PA317 cells expressing gPr80gag produced 5- to 10-fold more infectious vector or viral particles as well. Metabolic labeling studies indicated that this reflected enhanced virus particle release rather than increased viral protein synthesis. These results indicate that gPr80gag is important for M-MuLV replication in vivo and in vitro and that the protein may be involved in a late step in viral budding or release.

DNA vaccination against foot‐and‐mouth disease via electroporation: study of molecular approaches for enhancing VP1 antigenicity

Foot-and-mouth disease virus (FMDV) affects susceptible livestock animals and causes disastrous economic impact. Immunization with plasmid expressing VP1 that contains the major antigenic epitope(s) of FMDV as cytoplasmic protein (cVP1) failed to elicit full protection against FMDV challenge. In this study, mice were immunized via electroporation with four cDNA expression vectors that were constructed to express VP1 of FMDV, as cytoplasmic (cVP1), secreted (sVP1), membrane-anchored (mVP1) or capsid precursor protein (P1), respectively, to evaluate whether expression of VP1 in specific subcellular compartment(s) would result in better immune responses. Electroporation enhanced immune responses to vectors expressing cVP1 or P1 and expedited the immune responses to vectors expressing sVP1 or mVP1. Immunization of mice via electroporation with mVP1 cDNA was better than sVP1 or cVP1 cDNA in eliciting neutralizing antibodies and viral clearance protection. Vaccination with P1 cDNA, nonetheless, yielded the best immune responses and protection among all four cDNAs that we tested. These results suggest that the antigenicity of a VP1 DNA vaccine can be significantly enhanced by altering the cellular localization of the VP1 antigen. Electroporation is a useful tool for enhancing the immune responses of vectors expressing VP1 or P1. By mimicking FMDV more closely than that of transgenic VP1 and eliciting immune responses favorably toward Th2, transgenic P1 may induce more neutralizing antibodies and better protection against FMDV challenge.

Expression, purification and crystallization of a birnavirus-encoded protease, VP4, from blotched snakehead virus (BSNV).

Blotched snakehead virus (BSNV) is a member of the Birnaviridae family that requires a virally encoded protease known as VP4 in order to process its polyprotein into viral capsid protein precursors (pVP2 and VP3). VP4 belongs to a family of serine proteases that utilize a serine/lysine catalytic dyad mechanism. A mutant construct of VP4 with a short C-terminal truncation was overexpressed in Escherichia coli and purified to homogeneity for crystallization. Using the sitting-drop vapour-diffusion method at room temperature, protein crystals with two distinct morphologies were observed. Cubic crystals grown in PEG 2000 MME and magnesium acetate at pH 8.5 belong to space group I23, with unit-cell parameters a = b = c = 143.8 angstroms. Trigonal crystals grown in ammonium sulfate and glycerol at pH 8.5 belong to space group P321/P312, with unit-cell parameters a = b = 158.2, c = 126.4 angstroms.

Structural Polymorphism of the Major Capsid Protein of a Double-Stranded RNA Virus: An Amphipathic α Helix as a Molecular Switch

The infectious bursal disease virus T=13 viral particle is composed of two major proteins, VP2 and VP3. Here, we show that the molecular basis of the conformational flexibility of the major capsid protein precursor, pVP2, is an amphipatic alpha helix formed by the sequence GFKDIIRAIR. VP2 containing this alpha helix is able to assemble into the T=13 capsid only when expressed as a chimeric protein with an N-terminal His tag. An amphiphilic alpha helix, which acts as a conformational switch, is thus responsible for the inherent structural polymorphism of VP2. The His tag mimics the VP3 C-terminal region closely and acts as a molecular triggering factor. Using cryo-electron microscopy difference imaging, both polypeptide elements were detected on the capsid inner surface. We propose that electrostatic interactions between these two morphogenic elements are transmitted to VP2 to acquire the competent conformations for capsid assembly.

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

BackgroundFoot‐and‐mouth disease virus (FMDV) causes a severe livestock disease, and the virus is an interesting target for virology and vaccine studies.Materials and methodsHere 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.ResultsBoth 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.ConclusionsThis 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. Copyright © 2005 John Wiley & Sons, Ltd.

Nodamura Virus RNA Replication in Saccharomyces cerevisiae : Heterologous Gene Expression Allows Replication-Dependent Colony Formation

Nodamura virus (NoV) and Flock House virus (FHV) are members of the family Nodaviridae. The nodavirus genome is composed of two positive-sense RNA segments: RNA1 encodes the viral RNA-dependent RNA polymerase and RNA2 encodes the capsid protein precursor. A small subgenomic RNA3, which encodes nonstructural proteins B1 and B2, is transcribed from RNA1 during RNA replication. Previously, FHV was shown to replicate both of its genomic RNAs and to transcribe RNA3 in transiently transfected yeast cells. FHV RNAs and their derivatives could also be expressed from plasmids containing RNA polymerase II promoters. Here we show that all of these features can be recapitulated for NoV, the only nodavirus that productively infects mammals. Inducible plasmid-based systems were used to characterize the RNA replication requirements for NoV RNA1 and RNA2 in Saccharomyces cerevisiae. Induced NoV RNA1 replication was robust. Three previously described NoV RNA1 mutants behaved in yeast as they had in mammalian cells. Yeast colonies were selected from cells expressing NoV RNA1, and RNA2 replicons that encoded yeast nutritional markers, from plasmids. Unexpectedly, these NoV RNA replication-dependent yeast colonies were recovered at frequencies 10(4)-fold lower than in the analogous FHV system. Molecular analysis revealed that some of the NoV RNA replication-dependent colonies contained mutations in the NoV B2 open reading frame in the replicating viral RNA. In addition, we found that NoV RNA1 could support limited replication of a deletion derivative of the heterologous FHV RNA2 that expressed the yeast HIS3 selectable marker, resulting in formation of HIS+ colonies.

Genetic and phenotypic variations of isolates of shrimp Taura syndrome virus found in Penaeus monodon and Metapenaeus ensis in Taiwan.

Distinct Taura syndrome virus (TSV) isolates were found in Metapenaeus ensis (isolate Tw2KMeTSV), Penaeus monodon (isolate Tw2KPmTSV) and Litopenaeus vannamei (isolate Tw02LvTSV). Nucleotide sequence analysis of these three isolates revealed differences in the TSV structural protein (capsid protein precursor) gene orf2. TSV ORF2 amino acid sequence comparison and phylogenetic analysis suggested a comparatively close relationship between these three Taiwanese isolates and the Hawaiian isolate HI94TSV. In P. monodon specimens that were naturally and experimentally infected with the Tw2KPmTSV isolate, the virus was contained and shrimps showed no clinical signs of infection. However, when P. monodon was challenged with the Tw2KMeTSV isolate, the virus replicated freely. The ORF2 amino acid sequence of the Tw2KMeTSV isolate differed from that of isolate Tw2KPmTSV in four positions and these differences may account for their phenotypic differences, at least in terms of their ability to replicate in specific hosts.

Flock House virus subgenomic RNA3 is replicated and its replication correlates with transactivation of RNA2

The nodavirus Flock House virus has a bipartite genome composed of RNAs 1 and 2, which encode the catalytic component of the RNA-dependent RNA polymerase (RdRp) and the capsid protein precursor, respectively. In addition to catalyzing replication of the viral genome, the RdRp also transcribes from RNA1 a subgenomic RNA3, which is both required for and suppressed by RNA2 replication. Here, we show that in the absence of RNA1 replication, FHV RdRp replicated positive-sense RNA3 transcripts fully and copied negative-sense RNA3 transcripts into positive strands. The two nonstructural proteins encoded by RNA3 were dispensable for replication, but sequences in the 3′-terminal 58 nucleotides were required. RNA3 variants that failed to replicate also failed to transactivate RNA2. These results imply that RNA3 is naturally produced both by transcription from RNA1 and by subsequent RNA1-independent replication and that RNA3 replication may be necessary for transactivation of RNA2.

Flock House virus subgenomic RNA3 is replicated and its replication correlates with transactivation of RNA2

The nodavirus Flock House virus has a bipartite genome composed of RNAs 1 and 2, which encode the catalytic component of the RNA-dependent RNA polymerase (RdRp) and the capsid protein precursor, respectively. In addition to catalyzing replication of the viral genome, the RdRp also transcribes from RNA1 a subgenomic RNA3, which is both required for and suppressed by RNA2 replication. Here, we show that in the absence of RNA1 replication, FHV RdRp replicated positive-sense RNA3 transcripts fully and copied negative-sense RNA3 transcripts into positive strands. The two nonstructural proteins encoded by RNA3 were dispensable for replication, but sequences in the 3′-terminal 58 nucleotides were required. RNA3 variants that failed to replicate also failed to transactivate RNA2. These results imply that RNA3 is naturally produced both by transcription from RNA1 and by subsequent RNA1-independent replication and that RNA3 replication may be necessary for transactivation of RNA2.

Dissection of cauliflower mosaic virus transactivator/viroplasmin reveals distinct essential functions in basic virus replication.

Cauliflower mosaic virus (CaMV) transactivator/viroplasmin (Tav) is an essential multifunctional viral protein. Dissection of Tav by deletion mutagenesis revealed that the central region is essential for CaMV replication in single cells but that the N- and C-terminal parts are not. Strains with mutations in the central region were defective in the translational transactivator function and could be complemented by coexpressing Gag (capsid protein precursor) and Pol (polyprotein with protease, reverse transcriptase, and RNase H activity) from separate monocistronic plasmids. In contrast, total omission of Tav was only partially complemented by Gag and Pol overexpression from separate plasmids. These results indicate that CaMV basic replication requires both Tav-activated polycistronic translation and some posttranslational function(s) of Tav that is not affected by the deletions in the central region of Tav.

The cis-acting replication signal at the 3′ end of Flock House virus RNA2 is RNA3-dependent

The nodavirus Flock House virus has a bipartite positive-sense RNA genome consisting of RNAs 1 and 2, which encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively. The RdRp catalyzes replication of both genome segments and produces from RNA1 a subgenomic RNA (RNA3) that transactivates RNA2 replication. Here, we replaced internal sequences of RNAs 1 and 2 with a common heterologous core and were thereby able to test the RNA termini for compatibility in supporting the replication of chimeric RNAs. The results showed that the 3′ 50 nt of RNA2 contained an RNA3-dependent cis-acting replication signal. Since covalent RNA dimers can direct the synthesis of monomeric replication products, the RdRp can evidently respond to cis-acting replication signals located internally. Accordingly, RNA templates containing the 3′ termini of both RNAs 1 and 2 in tandem generated different replication products depending on the presence or absence of RNA3.

Recombinant adenovirus co-expressing capsid proteins of two serotypes of foot-and-mouth disease virus (FMDV): in vitro characterization and induction of neutralizing antibodies against FMDV in swine

Human adenovirus type 5 (Ad5) has been evaluated as a novel gene delivery vector for the development of live-viral vaccines for foot-and-mouth disease (FMD). In this study, we constructed an Ad5 vector co-expressing the capsid precursor proteins, P1, of FMD virus (FMDV) field strains A24 Cruzeiro and O1 Campos and examined the neutralizing antibody responses in swine after inoculation with the vector. To construct the Ad5 vector, a bicistronic expression cassette containing a cytomegalovirus promoter, the P1 coding sequence of FMDV A24, the internal ribosomal entry site (IRES) of FMDV A12, the P1 coding sequence of FMDV O1 Campos and the coding region of A12 3C protease was inserted into the E1 region of an E1/E3-deleted Ad5. The recombinant adenovirus, Ad5A24+O1, was generated by transfection of 293 cells with full-length pAd5A24+O1 recombinant plasmid DNA. The recombinant Ad5 co-expressed P1 of both A24 and O1 in infected 293 cells and P1 of both serotypes was processed to produce VP0, VP3, and VP1. We further demonstrated the formation of capsid protein complexes by co-precipitation of VP0, VP3, and VP1 with monoclonal antibodies against viral capsid proteins. Swine inoculated with Ad5A24+O1 generated neutralizing antibodies against both A24 and O1. However, the overall neutralizing antibody response was considerably lower than that induced by a commercial FMD vaccine or a monovalent Ad5-A24 vaccine.