Isolation And Identification Of Porcine Transmissible Gastroenteritis Virus LJ-12Strain

Transmissible gastroenteritis virus(TGEV) is a Pathogen of Transmissible gastroenteritis(TGE). The clinical symptoms of TGEV is vomiting and dehydration and diarrhea. Different agesare susceptible with highly infectious, less than two week old piglets, and its mortality rate couldreach100%. The disease is wide range prevalence and high rates of morbidity and death, is Chinaand other countries’ one of the important disease to pigs death, bringing significant economic lossin the pig industry. As with most viral disease prevention is the same, vaccine is the best way tocontrol its prevalence of TGE. But at the moment of the TGEV research institute are faced withdifficulty in separation, low titer and low yield. So hindered to TGE disease prevention. Thisexperiment by virus isolation, increasing virus content and the transmissible gastroenteritis vaccinestrain reserves laid a foundation for further research.Some feces was obtained from Hei Longjiang, and identification of nucleotide by RT-PCRamplification technology. Use TGEV S gene primer amplification gene sequences to a period ofabout480bp.Homology of the S gene position from TH-98strain is98.5%compared with thepublished in the GenBank.Show that onset pig diarrhea piglets TGE virus is contained in feces.Some feces from pigs with centrifugal filtration and inoculated into the primary pig kidney cellsand pig testis cell, they could produce prominence CPE by8generation of primary pig cells linesBy electron microscopy ultramicrotomy, negative staining electron microscopy, immune oelectronmicroscopy experiment found the virus have envelope and about80～100nm in diameter, namedTGEV LJ-12strains. The indirect immunofluorescence test and indirect ELISA test indicated theexpressive product had specific positive response with the separation strains transmissiblegastroenteritis virusat the eighth passage. Use TGEV S gene primer amplification gene sequencesto a period of about384bp by RT-nested-PCR amplification technology. Homology of the S geneposition compared with the published in the GenBank reached94%.Two pairs of specific primer was designed and according to the sequence of N gene of TGEVpublished in GenBank. Including outer primers P1/P2and inner primers N1/N2and inner primersN3/N4. P1/P2amplification length of1167bp.Using two pairs of primers were amplified byRT-nested-PCR. The results Suggests that the nucleotide sequence of N at length about475bp and476bp.Comparison of sequence by DNAStar,. Results show TGEV LJ-12strains withhomologous virus strains reached94%. The primary pig kidney cells cultures to20generation willstill be able to detect. Proved that the virus has separated successfully and has adapted to the primary cells.Animal experiments of the primary pig kidney cells cultures to15generation of cell culturevirus, Newborn not lactation SPF Bama pig gavaged with20mL cell culture virus; Experimentalpigs died in8days, Detection of feces and small intestine by RT–nested-PCR method.Thecorresponding target bars could be observed in RT-nested-PCR. Appear consistent with theexpected results of about475bp banding the N gene of purpose by inner PCR. Compared with cellculture virus gene sequence, sequence unchanged. By immunofluorescence detection method todetect the mesenteric lymph nodes, The corresponding target bars could be observed inRT-nested-PCR, Two test results are consistent, Shows experimental pigs are killed with a virus.Isolation successfully of TGEV LJ-12, For the epidemiological characteristics, laboratorydiagnosis and the biological characteristics of viruses and the transmissible gastroenteritis vaccinestrain reserves laid a foundation for further research.