The structure genes spike(S) ,nucleocapsid(N) ,membrane(M) ,small membrane(sM) of a porcine epidemic diarrhea virus(PEDV) strain DX isolated in Gansu province,North-west of China,were cloned,sequenced and compared with published sequences of PEDV strains. The nucleotide sequences encoding the entire S,sM,M and N genes open reading frame(ORF) of DX were 4 152,231,681 and 1 326 bases long respectively. There were transcription regulatory sequences(TRSs) upstream of the initiator ATG of the S,N and M genes. The amino acids sequences of S,M and N contained 30,3 and 7 potential asparagine(N) -linked glycosylation sites. Homologous analysis and phylogenetic trees showed that DX had the closest relationship with strains LJB/06,JS-2004-2Z and CH/HLJH/06 that were also isolated from China and indicated the prevalence of some PEDV isolates in China were widespread since the JS-2004-2Z strain originated from the south of the China,and LJB/06 and CH/HLJH/06 were isolated from northeast China. The N gene was cloned using two primers which contained Nco I and BamH I restriction enzyme sites and subcloned into expression vector pET30a. The recombinant plasmid was then transformed into E.coli Rossta. SDS-PAGE showed there was a protein of about 55kDa as expected and Western blot indicated the N protein had biological activity.
The complete genome sequence of transmissible Gastroenteritis virus (TGEV) strain TS, previously isolated from Gansu province, was cloned and compared with published sequence data from other TGEV strains. Phylogenetic tree analysis based on the amino acid and nucleotide sequences of the S gene showed that the TGEV strains were divided into 3 clusters. TGEV TS showed a close evolutionary relationship to the American Miller cluster but had a 5' non-translated region (NTR) sequence closely related to the American Purdue cluster. Continued culture in different cell types indicated that TGEV TS virulence could be attenuated after fifty passages in Porcine kidney (PK-15) cells, and that the Porcine kidney cell line IB-RS-2 (IBRS) was not suitable for culture of the TGEV strain TS.
