| Porcine epidemic diarrhea(PED) is a highly contagious, enteric disease of swine characterized by vomiting, dehydration, and a high mortality in piglets. The disease has been found in many countries including China , and causes economic losses in pig fanning.In the present study, an accurate diagnosis has been made for five pig farms that outbroke virus-associated diarrhea in pigs on the base of clinical diagnosis, PCR detection and virus isolation, and two of them were identified as PEDV infected farms. The results from the clinical data showed that the morbidity rates are 72.69% and 89.95% in suckled piglets, 68.07% and 75% in growing and fattening pigs, while the mortality rates are 62.88% and 81.31% in suckled piglets, 8.31% and 10.37% in other age groups respectively on the two farms. Although swine of all ages are susceptible to mis viral infection, the mortality in piglets under 4 weeks is commonly observed. The outbreaks usually occur during the winter as well as the early spring, and the major clinical signs can be observed as severe watery or yellowish diarrhea.Isolation and identification of virus were made on continuous Vero cells line. Fecal specimen, which are positive for amplifying M gene of PEDV with nested-PCR, were inoculated on the cells, and the blind passages were undertaken until the CPEs could be seen on 18 passages. The cells become rounded, lysed and off the wall of the vessels. For testifying the presence of PED isolate on Vero cells, RT-PCR technique was used to amplify M gene at the 5, 10, 15 and 20 passages., and a specific fragment of PEDV M gene was amplified, the result showed that the isolate has adapted to Vero cells.Two pairs of primers were designed according to the published M gene sequences of PEDV CV777 and Brl/87 strains with Oligo4.1 and Primer 5.0 software. A predicted 681-bp fragment of the M gene of PEDV was amplified from the RNA extracted from the fecal samples by RT-nested-PCR, and then cloned into the pMD18-T vector. The complete sequences of the M gene of the PED epidemic strain is 681 nucleotides, encodes 226 amino acids. The sequences analysis results indicated that the nucleotide sequence of PED epidemic strain has 98.09% homology in comparison with that of CV777, except for 13 nucleotide differences in the sites of 37, 47, 138, 179, 192, 213, 269, 285, 348, 529, 579, 618 and 629, and share 97.94% homology compared to Brl/87, except for 14 nucleotide differences in thesites of 37, 47, 138, 179, 192, 213, 269, 285, 348, 448, 529, 579, 618 and 629. The deduced animo acid homology is 97.79% and 98.23%, and there are differences in 13 and 14 animo acids as compared to CV777 and Brl/87, respectively. Comparison CV777 to Brl/87, they share 99.85% identities in nucleotide sequences, and 99.85% in deduced amino acid sequence. There is only one nucleotide difference between CV777 and Brl/87. All these data showed that there are some mutations in M gene of FED epidemic strain.Candidate: Lang Jing Hua Major: Preventive Veterinary ScienceTutor: Professor Li Yi Jing Professor QiBo...
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