Complete genome sequence analysis and construction full-length infectious cDNA clone of an epidemic Foot-and-mouth disease virus of serotype A

Abstract:

[Objective] Sequencing and analysis complete genome of an epidemic foot-and-mouth disease virus of serotype A and construction of its full-length infectious cDNA clone. [Methods] The primers were designed according to the published genome sequence of type A FMDV and a total of four fragments covering the complete genome of A/Sea-97/CHA/2014 were subsequently PCR amplified and sequenced. The four fragments were cloned into pBluescript SKhdv vector in turn with a single restriction sites to construct a full-length cDNA clone of A/Sea-97/CHA/2014 strain. The full-length plasmid linearized with Not Ⅰ and transfected to BSR/T7 cells expressing T7 RNA polymerase to rescue the recombinant virus. [Results] The result of sequence analysis showed that FMDV A/Sea-97/CHA/2014 strain was 8 171 bp in length [except poly(C) and poly(A)], which contains a 5′-UTR with 1 091 bp, a 3′-UTR with 95 bp and a ORF encoding 2 332 amino acids with 6 996 bp. The phylogenetic tree based on the nucleotide sequences of VP1 gene revealed that the A/Sea-97/CHA/2014 strain had a high sequence identity with A/GDMM/CHA/2013 strain (99.1%). The full-length plasmid transfected to BSR/T7 cells, apparent CPE were observed after 68 h incubation. The harvested virus was verified by IFA, RT-PCR and sequencing. The results indicated that infectious FMDV was successfully rescued in vivo. Plaque phenotype and growth curves tests of rescue virus and parental virus showed they have a similar growth phenotype and capacity of proliferation. [Conclusion] This study provided a useful material for studies of the FMD pathogen ecological distribution, molecular epidemiological as well as novel marker vaccines.