Isolation And Identification Of High Pathogenic PRRSV,Establishment Of RT-PCR Diagnosis Assay

An unparalleled large-scale of an originally unknown so-called"high fever"disease in China outbreak in 2006, which characterized by high fever, eye conjunctivitis, cough, asthma, neurological symptoms and high mortality. Because of serious influence of this disease to the national economy, it is very essential to diagnose, isolate and identify the causative agent.Artificial case in 30-day-aged pigs was made using the disease materials collected from Hubei province, isolated and identified was by some kinds of primary cells and cell lines,and tested TCID50, RT-PCR amplifyed the sequence of Nsp2 and the structural protein area.The pathological changes of the organs or tissues was observed under the microscope..1. Isolate on of the virus using the PAMs, pig kidney primary cells, Marc-145, PK-15 and BHK-21:it turned out that only the Marc-145 cells line had the clear CPE, and the CPE could reach 70% at 48h-72h after 3 passages of the virus, as well as TCID50 can reach 10-5.51/ml at 7th passage. So a high pathogenic PRRSV stain was abtained successfully (named HBGS). This isolation strain has a high pathogenicity which has passaged for 14th generation by now, and may be a good candidate strain for vaccine.2. Comparison of sequence of Nsp2 and structural protein area: A conclusion can be got that the homology between HBGS and the European standard strain LV stain, the American standard strain ATCC VR-2332 stain were 66.2% and 91.8% respectively, the homology between HBGS and CH1a, HB-1, BJ-4 were 96.2%, 97.1%, 91.6% respectively. The result above proves that the real causative agent was probably Chinese local varied PRRSV strain.3. Histological observation on lung, kidney, liver, heart, spleen and lymph:It showed that bronchopneumonia in lung cells, congestion and different degree of granular degeneration in liver cells, hemorrhage, congestion and large-scaled cells necrosis in spleen cells, glomerulonephritis in kidney cells, large-scaled cells necrosis and granular degeneration in heart cells. All of them manifested that the high pathogenic PRRSV can cause multisystem and multi-organ failure, of which the main lesion is in lung.In this research a sensitive detection assay have been designed based on ORF7 and Nsp2 which can set a concrete foundation for PRRS monitoring and molecular epidemiology investigation.