Nucleic Acids Research

Cloned cDNA molecules from three serotypes of FMDV have been sequenced around the VPl-coding region. The predicted amino acid sequences for VPl were compared with the published sequences and variable regions identified. The amino acid sequences were also analysed for hydrophilic regions. Two of the variable regions, numbered 129-160 and 193-204 overlapped hydrophilic regions, and were therefore identified as potentially immunogenic. These regions overlap regions shown by others to be immunogenic. INTRODUCTION Foot and mouth disease virus (FMDV) is a picornavirus causing a disease of domestic livestock which is of considerable economic importance (1). A complicating factor in control of the virus by vaccination is the existence of seven non-crossprotective serotypes:three European, A, O and C, three South African, SAT 1, SAT 2 and SAT 3, and one Asian, Asia 1. In addition each serotype is composed of a number of serologically related subtypes which are only partially cross-protective (2). With the development of recombinant DNA technology and rapid DNA sequencing, it is now possible to study in detail the extent of variation between the subtypes and serotypes. This is of particular relevance when applied to one of the major capsid polypeptides, VPl. This protein has been shown to be the major immunogen of the virus (3,4) and as such variation in its structure is likely to be responsible, at least in part, for the existence of the serotypes and subtypes. The complete primary structure (as predicted from the nucleotide sequence) of VPl has recently been reported for two subtypes of serotype A [A^Q61 (5) A12119 (6)] and one of serotype O [O^Kaufbeuren (O.K) (7)1 We report here the construction of recombinant plasmids containing cDNA coding for VPl for three more strains: A-^Cruzeiro, O. British Field Strain (O,BFS) and C,Indaial. Together with the previously published data, these results allow comparisons to be made between representatives of the three major European serotypes (A. .61, O.BFS and C, Indaial), between three subtypes of the same serotype (A.,.61, A,-119 and © IR L Press Limited, Oxford, England. 8285 0305-1048/82/1024-8285S 2.00/0 Nucleic Acids Research A-^Cruzeiro), and between two isolates of the same subtype (O,BFS and O,K). The conclusions on the distribution of the regions of variabil ity are discussed and compared with the recent results of Strohmaier et al (8) on the antigenicity of peptide fragments of VP1 from purified virus and those of Bit t le et al (9) and Pfaff et al (10) on the ability of short synthetic peptides, corresponding to parts of VP1, to elicit the production of neutralizing antibody. MATERIALS AND METHODS (a) Construction of recombinants Three strains of FMDV were used in this workc-A. ^Cruzeiro, C,Indaial and O,BFS. The FMDV RNA was reverse transcribed using either oligo dT (11) or calf thymus DNA oligonucleotides (12) as primer. [The calf thymus DNA primed reverse transcription would be expected to generate cDNA molecules randomly distributed throughout the FMDV genome as distinct from the oligo dT primed reverse transcription which has tended to bias the cDNA population to the 3' end (11)]. The resulting double stranded cDNA was inserted into the Pst I site of the plasmid pAT153 by G:C tailing as described by Boothroyd et al (11). The recombinant plasmids were used to transform competent E. coli HB101 cells. (b) Screening of recombinants The transformed cells were screened by the colony hybridization procedure described by Hanahan and Meselson (13). The C , and A_* recombinant plasmid transformed cells were probed with a Pvu II fragment of pFA. _t 76 (5) which contains DNA sequences derived from A,g61, covering most of the regions coding for VP3 and VP1. The O, transformed cells were probed with a small Rsa I/Hinfl fragment of pFO, t351 [Fig l (c) ] which was derived from the plasmid pFO, t251 (11), and comprises mainly the 3' end of the VP1 coding sequence. The positive colonies were further screened by restriction mapping of their plasmid DNA. These maps were aligned with a linear map of the protein coding units of the FMDV genome, by analogy with the cDNA sequences of A,g61 (5) of A ^ l l ^ (6) and of O,K (7). In some cases, Southern blotting of appropriate restriction fragments (14) was necessary to resolve ambiguities. (c) Sequencing Al l DNA sequencing was done by the method of Maxam and Gilbert (15) as described in Boothroyd et al (5). (d) Analysis of Sequence Data An Apple II microcomputer was used to search DNA sequences for restriction sites and to predict the corresponding protein sequences, using a programme writ ten by Larson and Messing (16). It was also used to make hydrophilicity plots