Cloning and sequence analysis of the M gene of porcine epidemic diarrhea virus LJB/03

Porcine epidemic diarrhea virus (PEDV) LJB/03 was isolated from the fece of piglets infected with PEDV on a pig farm, Heilongjiang province, China. The M gene of LJB/03 was amplified from the RNA extracted directly from the fece samples by RT-PCR and cloned into pMD18-T vector. The M gene cDNA was sequenced and encompasses an open reading frame of 681 nucleotides, encoding a 226-amino acid protein. The LJB/03 M gene has a base composition of 152 adenines (22%), 153 cytosines (23%), 161 guanines (24%), and 214 thymines (31%). Sequence comparison with other PEDV strains selected from GenBank revealed that the LJB/03 M gene has a high sequence homology to those of other PEDV isolates, 97.80% with JMe2, 96.92% with KPEDV-9 (Korean field isolate), 97.36% with KPEDV-9 (Korean), 97.80% with Br1/87, and 97.94% with CV777. The encoded protein shared 97.79% amino acid identities compared with CV777, 97.35% with Br1/87, 97.79% with JMe2, 96.90% with KPEDV-9 (Korean field isolate), 96.46% with KPEDV-9 (Korean). Sequence analysis of the M gene, including genetic distance measurement, phylogenetic tree analysis, and residue substitution analysis, showed that all other PED viruses analyzed fell into three groups, and the LJB/03 itself branched in an independent group. These data revealed that the M gene nucleotide sequence of LJB/03 has some mutations in comparison with the other PED viruses.