Isolation, Identification And Genome Sequence Of Porcine Enteric Virus

Objective: Porcine enteric virus is a pathogen, which causing porcine enterovirus infection, such as porcine encephalomyelitis, reproductive failure, pneumonia, diarrhea, cardiopericarditis, myocarditis, dermal lesion, and so on . It is an important infection disease that can damage the pig-raising industries. In this study, porcine enterovirus was isolated and identificated in pocine kidney primary cell.After the sequences alignment of the different serotypes of porcine enteric virus, we designed and synthesized the primers according to the published sequences of UKG 410/73(Y14459). At last we constructed a RT-PCR methods to differentiate the serotypes of porcine enteric virus, for the diagnosis and prevention provides the science the basis.Method: After suspending the field samples by phosphate-buffered saline, we cultured it in blind passage for several times in primary porcine kidney cell until the appearance of the cytopathic effect. Froze and thawed for there times, collected the cells, and then we found the virus by the electron microscope. According to the sequences of 5'untranslated region of porcine enteric virus, we designed the primers, got the gene, cloned it to the pMD-18T vector and sequenced. Then we analysized the sequence with other related sequences with DNAStar and NCBI BLAST definited the serotype. In this study, We also sequenced the complete sequences, and aligned it with thirty other sequences in order to analyse its change characteristic.Results: After blind passages for three times in porcine kidney primary cell, the cytopathic effect appeared: in the cell the pellet increased, the cell contracted and changed thecircle, at last the cell falled off from the bottle wall. Under the electron microscope, the virus was circular, icosahedral symmetry and had the non-pouch membrane. The 5'untranslated was amplified by RT-PCR. It is showed that the homology was very high with UKG 410/73(Y14459), showing 85.5% identity in nucleotide sequence, and combine immune the electron microscope, it was PEV-9. Based on the reported sequences of UKG 410/73(Y14459), eight pair of primers to amplify was designed. The sequences were aligned with 30 sequences from GenBank.It is showed that the homology with UKG 410/73(Y14459) was very high, showing 85.7% identity in nucleotide sequence and 86.6% identity in deduced amion acid sequence. In this study, we got its complete sequences and found that it was porcine enteric virus 9. It was prove that there are porcine enterovirus infection in our contry.