Isolation And Identification Of PRRSV And Effect Of PRRSV Isolate On Progesterone Secretion In Porcine Cycle Luteal Cells

Porcine reproductive and respiratory syndrome virus（PRRSV）infection can cause reproductive disorders related diseases such as abortion,stillbirth,mummified fetus,and infertility in sows.At present,vaccines cannot provide complete protection due to the continuous emergence of PRRSV mutants.Therefore,monitoring the infection of PRRSV is important for the prevention and control of the disease.Previous studies have shown that PRRSV can infect porcine ovaries.Ovarian corpus luteum is a temporary endocine organ,playing an important role in maintenance of estrous cycle and pregnancy.However,it is unclear whether PRRSV infection affects luteal function.Therefore,in the present study,samples were collected from a scaled pig farm and a native pig breeding farm detected PRRSV infection in a certain area of Hunan was isolated and identified.The effect of isolates on progesterone secretion of primary porcine ovarian luteal cells was further studied.The results of this study are as follows:1.Isolation,identification,and genetic evolution analysis of PRRSV（1）A total of 330 pig tissue and serum samples were collected from large-scale farms,harmless treatment farms,slaughterhouses in a certain area,and 287 whole blood samples were collected from local pig breeding farms in in Hunan province.The pathogen of PRRSV was detected by fluorescence quantitative PCR.The results showed that PRRSV nucleic acid could be detected in the samples with a positive rate of 10.3%（34/330）in the large-scale farm and 2.1%（6/287）in the native breeding farm,respectively.（2）Further genetic evolution analysis was carried out by amplifying ORF5gene from the PRRSV positive samples of the native breeding farm.The results showed that six ORF5 gene sequences were obtained,all of which belonged to the lineage 8branch of PRRSV-type 2,sharing a similarity of 99.2%to 99.8%between each strains,and 83.1%to other PRRSV-2 strains at home and abroad.There are differences in the amino acid sequence of the coded GP5 in signal peptide region（the 23rd position）,potential N-glycosylation site（the 33rd position）and epitope C（the 59th position）.（3）The positive serum was inoculated to Marc-145 cells and passed blindly,and obvious cytopathic effect appeared after the fifth passage.A strain of PRRSV was isolated and identified,designated as PRRSV NX-1,and its titer was 4×10⁵TCID₅₀/m L2.Effect of the PRRSV isolate on progesterone secretion of luteal cells.（1）Porcine ovarian corpus luteum was collected and digested with type II collagenase.The 3β-HSD staining showed that the purity of isolated luteal cells was more than 90%.（2）Luteal cells were infected with isolated strain with MOI 0.1 for 24,48 and 72 h.The expression of PRRSV ORF7 in porcine luteal cells was detected by absolute fluorescence quantitative PCR.The results showed that the expression of ORF7 gene in luteal cells significantly increased with the development of infection time（P<0.05,P<0.01）.（3）CCK-8 assay were used to detect the viability of PRRSV-infected luteal cells.Cells was infected with MOI 0.1 or 1 for 24,48 and 72 h.The results showed that the cell viability was significantly increased in a dose-and time-dependent manner after virus infection（P<0.05,P<0.01）.The cellular proteins were extracted at 48 h after infection.The western blotting assay showed that the expression of the proliferating cell nuclear antigen（PCNA）was significantly increased,and the ratios of p-ERK1/2/ERK1/2 and p-p38/p38 increased as well,but the ratio of p-JNK/JNK decreased significantly.（4）Chemiluminescence assay showed that the progesterone level was significantly increased in the cell culture medium when the luteal cells was infected with 1 of MOI PRRSV for 48 h,when compared to that of the control cells.The present data showed that there was PRRSV infection in both large-scale pig farm and native pig breeding farm.Six PRRSV ORF5 gene sequences obtained from native PRRSV positive samples belonged to PRRSV-2 with many variations in GP5amino acids.The isolated PRRSV strain promoted the cell proliferation and progesterone secretion of porcine luteal cells,which may regulate the cell functions through the MAPK pathway,and its specific mechanism needs further study.