Primary structure of the DNA copy of the protein VP1 gene of the foot-and-mouth disease virus A22

The DNA-copy of the major antigen (VP1) coding region of the FMDV A22 sero-type has been cloned and sequenced. A comparison of the respective amino acid sequence with those of other VP1 of A-serotype revealed considerable differences in the structure of antigenic determinants.     

A new approach to DNA synthesis from synthetic oligonucleotides. Synthesis of DNA coding for the repeated antigenic determinant of foot and mouth disease virus

A rapid method for assembly of DNA from synthetic oligodeoxynucleotides has been developed which involves separate ligation of top- and bottom-strand oligonucleotides followed by filling in 3'-ends of the duplex formed, blunt end cloning into a specialized vector pBBV, and recovery of the synthetic DNA from the recombinant plasmid by means of restriction nuclease BbvII. The method allows for many oligonucleotides to be ligated at once, with no intermediates being isolated, and any DNA to be recovered on cloning, no matter what the sequences of its termini are. Ten oligodeoxynucleotides (I)-(X) have been chemically synthesised and used to prepare, by this method, a 60-membered duplex with complementary tetranucleotide 5'-protrusions (DNA I) which comprises the cDNA sequence 3397-3456 of foot and mouth disease virus (FMDV) strain O1K. Self-ligation of the duplex in the head-to-tail manner yielded 120 to 900 bp long synthetic DNAs (DNA II-DNA XV) coding for oligomers of the major antigenic determinant (the amino acid sequence 141-160 of protein VP1) of FMDV. The synthetic hexamer (DNA VI) was fused to gene lacZ' on plasmid pBBV21 and expressed in E. coli. The fusion was found to complement the lacZ deletion M15, from which it follows that the fused protein associated with the alpha-deficient beta-galactosidase to yield a tetramer carrying, on its N-termini, 24 antigenic determinants of FMDV.     

Correlation between efficacy and structure of recombinant epitope vaccines against bovine type O foot and mouth disease virus

To develop recombinant epitope vaccines against foot-and-mouth disease virus (FMDV), genes coding for six recombinant proteins (rP1–rP6) consisting of different combinations of B cell and T cell epitope from VP1 capsid protein (VP1) of type O FMDV were constructed and the 3D structure of these proteins analyzed. This revealed a surface-exposed RGD sequence of B cell epitopes in all six recombinant proteins as that in VP1 of FMDV and rP1, rP2 and rP4 globally mimicked the backbone conformation of the VP1. rP1, rP2 and rP4 stimulated guinea pigs to produce higher level of neutralizing antibodies capable of protecting suckling mice against FMDV challenge. rP1 stimulated cattle to produce FMDV-neutralizing antibody. The data suggest that an efficient recombinant epitope vaccine against FMDV should share local similarities with the natural VP1 of FMDV.     

Nucleotide sequence and corresponding amino acid sequence of the gene for the major antigen of foot and mouth disease virus

A segment of 1160 nucleotides of the FMDV genome has been sequenced using three overlapping fragments of cloned cDNA from FMDV strain O1K. This sequence contains the coding sequence for the viral capsid protein VP1 as shown by its homology to known and newly determined amino acid sequences from this man antigenic polypeptide of the FMDV virion. The structural gene for VP1 comprises 639 nucleotides which specify a sequence of 213 amino acids for the VP1 protein. The coding sequence is not flanked by start and stop codons which is consistent with the mode of biosynthesis of VP1 by post-translational processing of a polyprotein precursor.     

Asia1型口蹄疫病毒分离株结构蛋白基因的克隆与表达

口蹄疫病毒结构蛋白氨基酸的变化是病毒抗原性变异的分子基础,大部分抗原表位位于主要的免疫原蛋白VP1上,部分非线性抗原表位位于VP2和VP3上。本研究首次成功测定了 Asia1 型口蹄疫病毒(YNBS/58)四种结构蛋白基因( p1 区)的核苷酸序列,全长 2199 个碱基,编码 733 个氨基酸,该基因与 Ind63/72、Pka3/54、Israel、China/99、C1/Germany、A22、ZIM7/83/2 毒株的 p1 基因核苷酸序列同源性分别为 88. 4%、86. 0%、89. 3%、68.6%、67.6%、66.8%、50.3%,推导的氨基酸序列同源性分别为 94.1%、93.2%、95.1%、79.9%、77.0%、76.5%、58.1%;将YNBS/58株与 Ind63/72、Pka3/54、Israel株的 vp1、vp2、vp3、vp4 基因和编码蛋白分别进行同源性比较,发现VP1的序列变异最大,VP2、VP3、VP4次之,且VP1的氨基酸变异主要集中在 42-50 位和 137-156 位。实现了YNBS/58株结构蛋白基因在大肠杆菌中的高效表达,其表达的融合蛋白以包涵体形式存在,分子量约为88kDa,占菌体总蛋白的16%左右,并利用镍柱对目的蛋白进行了纯化,纯度达 90%以上,本实验为进一步研究 A sia1型口蹄疫病毒的分子流行病学、p1基因及其编码蛋白的生物学功能奠定了基础。     

Effects of amino acid substitutions in the VP2 B-C loop on antigenicity and pathogenicity of serotype Asia1 foot-and-mouth disease virus

ABSTRACT: BACKGROUND: Foot-and-mouth disease virus (FMDV) exhibits a high degree of antigenic variability. Studies of the antigenic diversity and determination of amino acid changes involved in this diversity are important to the design of broadly protective new vaccines. Although extensive studies have been carried out to explore the molecular basis of the antigenic variation of serotype O and serotype A FMDV, there are few reports on Asia1 serotype FMDV. METHODS: Two serotype Asia1 viruses, Asia1/YS/CHA/05 and Asia1/1/YZ/CHA/06, which show differential reactivity to the neutralizing monoclonal antibody (nMAb) 1B4, were subjected to sequence comparison. Then a reverse genetics system was used to generate mutant versions of Asia1/YS/CHA/05 followed by comparative analysis of the antigenicity, growth property and pathogenicity in the suckling mice. RESULTS: Three amino acid differences were observed when the structural protein coding sequences of Asia1/1/YZ/CHA/06 were compared to that of Asia1/YS/CHA/05. Site-directed mutagenesis and Immunofluorescence analysis showed that the amino acid substitution in the B-C loop of the VP2 protein at position 72 is responsible for the antigenic difference between the two Asia1 FMDV strains. Furthermore, alignment of the amino acid sequences of VP2 proteins from serotype Asia1 FMDV strains deposited in GenBank revealed that most of the serotype Asia1 FMDV strains contain an Asn residue at position 72 of VP2. Therefore, we constructed a mutant virus carrying an Asp-to-Asn substitution at position 72 and named it rD72N. Our analysis shows that the Asp-to-Asn substitution inhibited the ability of the rD72N virus to react with the MAb 1B4 in immunofluorescence and neutralization assays. In addition, this substitution decreased the growth rate of the virus in BHK-21 cells and decreased the virulence of the virus in suckling mice compared with the Asia1/YS/CHA/05 parental strain. CONCLUSIONS: These results suggest that variations in domains other than the hyper variable VP1 G-H loop (amino acid 140 to 160) are relevant to the antigenic diversity of FMDV. In addition, amino acid substitutions in the VP2 influenced replicative ability and virulence of the virus. Thus, special consideration should be given to the VP2 protein in research on structure-function relationships and in the development of an FMDV vaccine.     

Synthesis of fusion proteins with multiple copies of an antigenic determinant of foot-and-mouth disease virus

A series of four expression plasmids coding for fusion proteins containing foot-and-mouth disease virus (FMDV) sequences was constructed. The fusion proteins contain a large part of beta-galactosidase from Escherichia coli preceded (N-terminal) by 1, 2, 4 or 8 repeats of the antigenic determinant of FMDV consisting of amino acids 137-162 of the capsid polypeptide VP1. All four fusion proteins were efficiently produced in E. coli host bacteria. Immunization of rabbits resulted in FMDV-specific, neutralizing antibodies, the response being dependent on the number of repeats. With enzyme-linked immunosorbent-assay techniques it was shown that the FMDV antigenic determinants are exposed on the surface of the fusion proteins under non-denaturing conditions.     

Recombinant plasmids containing hybrid protein genes with antigenic determinants of the foot and mouth virus

Plasmids have been constructed which contain genes coding for fused proteins including beta-galactosidase or human leukocyte interferon alpha 2 and monomeric or pentameric form of the main antigenic determinant of the foot-and-mouth disease virus (FMDV) serotype 01K. Expression of the hybrid genes has been studied. It is shown that fused proteins, containing beta-galactosidase and the antigenic determinant (monomer or pentamer), interact specifically with anti-FMDV anti-sera and with antibodies against peptide 141-160 of FMDV VP1 coat protein.     

Antigenic structure of the foot-and-mouth virus. V. Protection of naturally susceptible animals from foot-and-mouth disease using a synthetic peptide

We have synthesized the peptide representing 135-159 VP1 sequence of A22 strain of the foot-and-mouth disease virus (FMDV). The synthetic peptide induced 100% protection of guinea pigs against the disease. Two-fold immunization of cuttle with the peptide and single immunization of sheep induced full protection of the animals against A22 strain of FMDV.     

Detection of foot-and-mouth virus antibodies using a purified protein from the high-level expression of codon-optimized, foot-and-mouth disease virus complex epitopes in Escherichia coli

A codon optimized DNA sequence coding for foot-and-mouth disease virus (FMDV) capsid protein complex epitopes of VP1 amino acid residues 21-40, 135-160, and 200-213 was genetically fused to the C-terminal end of a glutathione-S-transferase (GST) gene in pGEX-6P-1 vector with the synonymous codons preferred by Escherichia coli . The gene was synthesized using PCR and subsequently expressed in E. coli producing an intracellular, soluble fusion protein that retained antigenicity associated with FMDV antibodies by western blot analysis. The chimera was purified from bacterial lysates by affinity chromatography and could be used in ELISA tests for antibodies against FMDV.     

A model of the secondary structure and distribution of antigenic determinants in the VP1 protein of hepatitis A virus

A comparative analysis of amino acid sequence of the proteins VP1 of hepatitis A virus and poliovirus of the 1 type was carried out. A model is proposed of structural organization of VP1 of hepatitis A virus providing the presence of a bilayer core formed by 8 antiparallel beta-strands. Probable candidates for surface antigenic determinants are the amino acid sequences located in unordered fragments of the polypeptide chain (residues 101-106 and 115-125), and alpha-helical region (residues 127-135).     

Bacterially expressed antigenic peptide from foot-and-mouth disease virus capsid elicits variable immunologic responses in animals

A fusion protein consisting of beta-galactosidase (GZ) to which was attached at its N-terminus the amino acid sequence corresponding to residues 142-160 of the immunogenic protein VP1 of foot-and-mouth disease virus (FMDV) has been expressed in E. coli. A chemically synthesized section of DNA corresponding to the amino acid sequence 142-160 was inserted into a vector (pXY410) designed to express fusion proteins with the carboxy terminal 1015 amino acids of GZ. The hybrid protein immunopurified by a GZ-specific monoclonal antibody was soluble, retained full GZ activity, and induced virus-neutralizing antibody in guinea pigs and mice. There were significant differences between the responses of individual mice to the FMDV peptide sequence, although the titers against GZ were uniformly high. This variable pattern did not change after hyperimmunization and was demonstrable in a range of mouse strains of different haplotype. The same results were obtained whether the response was measured by virus neutralization or by RIA against the FMDV peptide sequence. The possible reasons for the variable recognition of the FMDV epitopes by individual mice are discussed.     

Antigenic structure of the foot-and-mouth disease virus. II. Synthesis of protective peptides from the major immunogenic region of VP1 protein of foot-and-mouth disease virus type A22

Earlier we found that the immune response and antiviral protection from FMDV can be achieved by immunization with uncoupled FMDV peptides. In a search of approaches to animal protection from FMDV A22 strain we prepared a series of peptides corresponding to the putative antigenic determinants. Synthetic 131-149 and 140-149 sequences afforded 50 to 80% protection, both in the free state and conjugated with keyhole limpet hemocyanin. We believe that the 140-149 segment is so far the smallest peptide capable of eliciting specific antiviral protection without conjugation with a high molecular carrier.     

Serotype and VP1 gene sequence of a foot-and-mouth disease virus from Hong Kong (2002)

The nucleotide sequence of the VP1 coding region of foot-and-mouth disease virus (FMDV) strain HKN/2002, isolated from a disease outbreak occurring in Hong Kong in February 2002, was determined and compared with the sequences of other FMDVs. The VP1 coding region was 639 nucleotides in length and encoded a protein of 213 amino acid residues. Comparison of the VP1 nucleotide sequence with those of other isolates indicated that HKN/2002 belonged to serotype O. A VP1-based sequence similarity tree of several South-east Asian FMDV-O isolates showed that HKN/2002 was most closely related to FMDV isolates found in Hong Kong from 1991 to 1999 and Taiwan in 1997. Comparison of the amino acid sequence of the major immunogenic region of HKN/2002 with that of the serotype O vaccine strain, O1/Manisa/Turkey/69, reveals significant similarity, indicating that current serotype O vaccines may offer some degree of protection against HKN/2002.     

Evolution of the capsid protein genes of foot-and-mouth disease virus: antigenic variation without accumulation of amino acid substitutions over six decades.

The genetic diversification of foot-and-mouth disease virus (FMDV) of serotype C over a 6-decade period was studied by comparing nucleotide sequences of the capsid protein-coding regions of viruses isolated in Europe, South America, and The Philippines. Phylogenetic trees were derived for VP1 and P1 (VP1, VP2, VP3, and VP4) RNAs by using the least-squares method. Confidence intervals of the derived phylogeny (significance levels of nodes and standard deviations of branch lengths) were placed by application of the bootstrap resampling method. These procedures defined six highly significant major evolutionary lineages and a complex network of sublines for the isolates from South America. In contrast, European isolates are considerably more homogeneous, probably because of the vaccine origin of several of them. The phylogenetic analysis suggests that FMDV CGC Ger/26 (one of the earliest FMDV isolates available) belonged to an evolutionary line which is now apparently extinct. Attempts to date the origin (ancestor) of the FMDVs analyzed met with considerable uncertainty, mainly owing to the stasis noted in European viruses. Remarkably, the evolution of the capsid genes of FMDV was essentially associated with linear accumulation of silent mutations but continuous accumulation of amino acid substitutions was not observed. Thus, the antigenic variation attained by FMDV type C over 6 decades was due to fluctuations among limited combinations of amino acid residues without net accumulation of amino acid replacements over time.     

Asia Ⅰ型口蹄疫病毒中和表位与羊IgG重链恒定区的融合表达及免疫原性测定

VP1蛋白是口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)诱导机体产生抗病毒感染免疫的主要蛋白,含有病毒的若干中和表位.本研究设计和合成了由Asia Ⅰ型FMDV VP1蛋白136～160aa和198～211aa两个表位组成的重复串联表位的编码基因,并克隆了羊IgG重链恒定区编码基因.利用BamH I、EcoR I和Xho I位点将2个基因片段依次克隆到pPROExHTb载体,构建成重组质粒pPRO-FshIgG,将其转化大肠杆菌BL21(DE3)感受态细胞,以IPTG诱导表达得到融合蛋白FshIgG.100μg FshIgG蛋白免疫豚鼠后刺激豚鼠产生了高效价的FMDV中和抗体,而且使这些免疫豚鼠在用200 ID_(50)剂量FMDV攻击时得到了完全保护.由此证明,羊IgG重链恒定区蛋白能够作为FMDV表位肽的载体,而融合蛋白FshIgG可成为一种口蹄疫表位疫苗候选物用于口蹄疫的预防.     

Antibodies against a preselected peptide recognize and neutralize foot and mouth disease virus.

A major antibody combining site on foot and mouth disease virus (FMDV) serotype O1K has been identified in a predicted surface helix of viral protein 1 (VP1) between amino acid residues 144 and 159. A hexadecapeptide covering this sequence elicits high titers of antibodies that specifically recognize and neutralize FMDV. The high quality of the immune response is attributed to a particularly stable conformation of the antigenic amino acid sequence, which is most likely an alpha-helix.     

The molecular basis of the antigenic variation of foot-and-mouth disease virus.

We have cloned and sequenced the viral protein (VP1)-coding regions of two foot-and-mouth disease virus (FMDV) serotypes (C1 and A5). Comparison of the derived amino acid sequences with the known VP1 sequence of FMDV O1K and the two FMDV A subtypes A10 and A12 shows two highly variable regions in the protein, at positions 40-60 and 130-160, as possible antigenic sites. In both variable regions, several sites could be detected where all three sequences of the A subtypes are identical but the three types A, C and O differ from each other. The second variable region overlaps with a major immunogenic determinant of the virus.     

Analysis of neutralizing epitopes on foot-and-mouth disease virus.

For the investigation of the antigenic determinant structure of foot-and-mouth disease virus (FMDV), neutralizing monoclonal antibodies (MAbs) against complete virus were characterized by Western blot (immunoblot), enzyme immunoassay, and competition experiments with a synthetic peptide, isolated coat protein VP1, and viral particles as antigens. Two of the four MAbs reacted with each of these antigens, while the other two MAbs recognized only complete viral particles and reacted only very poorly with the peptide. The four MAbs showed different neutralization patterns with a panel of 11 different FMDV strains. cDNA-derived VP1 protein sequences of the different strains were compared to find correlations between the primary structure of the protein and the ability of virus to be neutralized. Based on this analysis, it appears that the first two MAbs recognized overlapping sequential epitopes in the known antigenic site represented by the peptide, whereas the two other MAbs recognized conformational epitopes. These conclusions were supported and extended by structural analyses of FMDV mutants resistant to neutralization by an MAb specific for a conformational epitope. These results demonstrate that no amino acid exchanges had occurred in the primary antigenic site of VP1 but instead in the other coat proteins VP2 and VP3, which by themselves do not induce neutralizing antibodies.     

Nucleotide sequence of the gene encoding the serotype-specific antigen of human (Wa) rotavirus: comparison with the homologous genes from simian SA11 and UK bovine rotaviruses

The nucleotide sequence of human (Wa) rotavirus genome segment 9, which encodes the serotype-specific antigen VP7, has been determined. Comparison of the deduced amino acid sequence of Wa VP7 protein to the sequences of simian SA11 and UK bovine VP7 proteins shows that the majority of the amino acid differences are clustered between amino acid residues 37 through 49, 65 through 75, 87 through 105, 122 through 126, 146 through 149, 178 through 181, and 208 through 242. A hydrophilicity profile of the three proteins reveals correlations between hydrophilic peaks, potentially antigenic determinants, and certain clusters of amino acid changes.