Isolation And Identification Of Porcine Epidemic Diarrhea Virus NJ Strain

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is an acute, highly contagious, and devastating enteric disease that is characterized by severe enteritis and diarrhea, with high mortality rates in suckling pigs. PED has spread and become widely prevalent in some countries and regions. Vaccine is the best way to control its prevalence of PED. However, PEDV difficult to adapt to the cells, virus titer degree is low, culture conditions is complex, has been the important problem of PEDV reproduction, limits the PEDV molecular biology research, seriously hindered the PED prevention. Therefor, according to the genetic variability of epidemic strains in our country, isolate and obtain PEDV strain adapted cells, has an important practical significance for the development of PED vaccine.To prepare a large number of PEDV adapted cells and improve viral titer, we isolate and propagate PEDV in IEC cell, porcine kidney cells and small intestine tissue cell cultures. We got an isolated PEDV NJ strain identified by morpha characteristic, molecular biological detection, immunodiagnosis and animal test, which provide a new method to propagate PEDV in cell line. This study provided a foundation to epidemiological characteristics, laboratory diagnosis, the biological characteristics of viruses for further research.The isolation and identification of PEDV NJ. Intestinal tract was infected with PEDV detected by RT-PCR and made into homogenate suspension, which was cultured in IEC, porcine kidney cells and small intestine tissue, named NJ strain. The generation of cell culture virus was45,28and3. The infected cells had occured CPE regularly. The three kinds of cells culture virus were able to detect by RT-nested-PCR amplification technology. Electron microscopy and immune electron microscopy experiment found the virus have envelope and about100-160nm in diameter, surface projections characteristic of coronavirus were evident Electron microscopy ultramicrotomy showed virus particles are spherical or oval-shaped, the virion diameter is about60-80nm. Some of virus particles were hollow, most were dense. Virus particles piled up in the cytoplasm. The indirect immunofluorescence test showed that expressive product had specific positive response with PEDV cells culture separately. Indirect ELISA detected the30and40generation of IEC-2cell culture virus, the10and20generation of kidney cell culture virus, the amount of virus increased gradually, the growth of kidney cell virus culture was obvious. Virus titration was performed by IEC cell culture virus. Titer of the virus rised gradually, reached10-4.5 TCID50/0.1mL at passage level45. Animal experiment showed that piglet appeared mild diarrhea after20h, watery diarrhea at4th day and died at6th day. The autopsy showed significant association with expansion, full of yellow liquid, intestinal wall thinning, mesenteric funicular hyperemia. Detection of feces and small intestine by RT-nested-PCR method. The corresponding target bars could be observed in RT-nested-PCR, comparing with cell culture virus gene sequence, sequence unchanged. The indirect immunofluorescence test of small intestine tissue was positive.These results indicated that the piglet infected PEDV NJ.The main sequences analysis of PEDV NJ. In order to analyze PEDV NJ genetic variability, the structure gene and ORF3sequence analysis of parent NJ was detected. The NJ strain has far relationship with vaccine strain and traditional strain, and high identity with the Chinese epidemic strains. The M gene of IEC cell culture virus at33passage level showed the gene changed a lot compared with parent NJ, divied into two groups, has a near relationship with attenuated strains DR13(Attenuated) and83P-5(100th). We amplified partial sequence of M gene from the parent, the5th,10th,15th and25th passaged NJ. Compared with the parent NJ, the gene changes occuring at the5th passage, and the mutations occuring at the15th passage was strongly. However, with the serial passage of the virus, the mustational gene at25th passage disappeared.Isolation of PEDV NJ successfully, provided an important material foundation for the basis of biological characteristics PEDV isolated virus and virus-related applied research.