Gene for the major antigenic protein of foot and mouth desease virus, type ASIA 1 63/72

Abstract

Foot and Mouth Disease Virus (FMDV) contains a positivesense singlestranded RNA enclosed in a protein capsid composed of four structural proteins: VP1, VP2, VP3, and VP4. VP1 is the major antigenic protein, and variations in this protein have resulted in the occurrence of seven serotypes and over sixty subtypes of the virus. India is a habitat for four serotypes: O, A, C and Asia 1. Serotypes O, A, and C have been extensively studied to develop costeffective and safer immunogens and to understand the molecular basis of antigenic variations. In order to understand the variations and to develop a vaccine against the Asia 1 virus, an isolate, 63/72, of the serotype Asia 1 was investigated. Doublestranded cDNA for the viral RNA was synthesized and inserted at the PstI site in the vector pUR222 by the dC/dG tailing method. The recombinant plasmids were transferred into E. coli RR1M15 and grown on plates containing 5Bromo4chloro3indolylDgalactopyranoside (Xgal) and isopropylthiogalactopyranoside (IPTG). The presence of the insert in colourless colonies was detected by colony hybridization using ³²Pendlabelled FMDV Asia 1 RNA. Colonies exhibiting strong hybridization signals were tested for production of VP1 by ELISA using antibodies raised against inactivated virus. The clone showing the strongest reddishbrown colour reaction was grown in the presence of ³Smethionine and the protease inhibitor phenylmethylsulphonyl fluoride. The labelled proteins were analysed by SDSPAGE. An additional protein band of 38 kDa was observed on the autoradiogram from the clone containing the insert cDNA. The labelled proteins were immunoprecipitated with monoclonal antibodies, antiVP1 antibodies, and antibodies against inactivated virus and analysed by SDSPAGE. A band corresponding to 20 kDa, in addition to the 38 kDa band, was observed. The presence of major antigenic epitopes of VP1 on both 38 kDa and 20 kDa proteins was confirmed by competitive ELISA, radioimmunoassay, and plaqueinhibition assay. Immunoprecipitates from antisera raised against proteins in the lysate showed an 18 kDa protein in addition to the 38 and 20 kDa antigenic proteins. The primary product of the cloned cDNA may be the 38 kDa protein, which undergoes proteolysis to yield the 20 and 18 kDa proteins. The proteins produced by the cloned cDNA were highly immunogenic in guinea pigs and cattle. With a single injection of 100 ng of the protein (equivalent to 4 µg of VP1), serum antibody titers in guinea pigs increased 16-32 fold (virusneutralization assay). A single injection of 10-20 µg of the protein protected 5 of 8 guinea pigs against a challenge dose of 100 guineapig ID of virulent Asia 1 virus. In cattle, 3 mg of lysate protein (equivalent to 120 µg of immunogen) injected on days 0 and 28 led to a 100fold increase in antibody levels within 40 days. Three of the eight immunized cattle were completely protected when challenged with 10,000 cattle ID of the virulent Asia 1 virus. The inserted cDNA coding for the antigenic protein was 0.9 kb in size. The insert contained PstI and SalI restriction sites; BamHI, HindIII, EcoRI, BglI, KpnI, SacI and HinfI sites were absent. Different restriction fragments were cloned into M13mp11 or M13mp19, and the recombinant phages were sequenced by the dideoxy chaintermination method. An 879nucleotide sequence was assembled from overlapping reads. The SalI and PstI sites were located at 483 and 733 nucleotides downstream of the 5 end. Alignment using the GAP program showed maximum homology (40%) with the VP1 gene of the C3 virus strain. The insert was 65% GCrich and lacked its own transcription and translation initiation signals. Expression was achieved via the lac promoter and AUG codon of the complementation region of galactosidase encoded by the vector. The insert was inframe with 5 amino acids at the Nterminus of the fragment. A polypeptide of 293 amino acids was deduced from the insert sequence. Its molecular weight was 31.6 kDa; including the 5 Nterminal amino acids (0.57 kDa) and 55 Cterminal vectorencoded amino acids (6.05 kDa), the expressed protein had a size of 38.2 kDa, matching the observed 38 kDa protein. The derived protein contained 43 arginine, 4 lysine, 7 glutamic acid, and 18 aspartic acid residues, making it highly basic-consistent with the biochemical properties of the 38 kDa immunogen. The cloned cDNA may serve as a probe for serotyping and for vaccine development against FMDV Asia 1 (63/72).