Fluorescence In Situ Hybridization Technique For Detection Of Porcine Reproductive And Respiratory Syndrome Virus And The Clinical Application

Porcine reproductive and respiratory syndrome virus, PRRSV is characterized by the respiratory disease of sucking pigs and the reproductive failure in sows, which generate huge economic losses. PRRSV is a single-stranded RNA virus, and has been classified in Arterivirus group, the genome of PRRSV contains 9 open reading frames(ORFs).The viruses are divided in to two groups: North American(VR-2332)and European(LV) isolates. In 2006,an outbreak of PRRS occurs in China, which also known as ’pig high fever disease’, like no other time before, the virus showed a high virulence, and the disease was characterized by high fever, high pathogenicity and high mortality. gene analysis indicates that the new virus belongs to VR-2332,named HP-PRRSV. Compare with classical IBDV, there is a nucleotides deletion in Nsp2 sequence of HP-PRRSV, about 90 bases, and can express stably.In the experiment, piglets were infected with Hu N4 virus by nasal drip. On days5,7,14 after infection, the piglets were anesthetized and killed,perfused with 4% paraformaldehyde and embed with paraffin. Observed the histopathologic changes and the distribution of PRRSV by Hematoxylin-eosin staining(HE)and lmmunofluorescent histochemical staining method. Analysis the sequence on Nsp2,design and synthesis fluorescent labeled oligonucleotide probe to test the samples by FISH, and investigate 50 case from different regions in Hei Longjiang province。The results show that the lesion appears first in lymph nodes and lungs. Serious Pulmonary congestion and oedema can be observed, presenting with consolidation and necrosis. Lymph nodes were edematous to touble to tenfold compared with the normal ones.Varying degrees of hemorrhage happens in the liver and spleen. Kidney swell or atrophy, petechia can be observed. Histologically observation shows that the most pathological changes were interstitial pneumonia. The number of lymphocytes in the lymph node were significantly decreased and necrosis. Large numbers of macrophage and mononuclear cell and inflammatory cell infiltration can be observed. The regularity of virus distribution can be viewed by immunofluorescence histochemistry, he result shows that the positive signal of virus can be detected in the lymph node、lungs and spleen at the 5d of infection;A t the 7d of infection, positive signal appears in the kidney、liver, but not intensity. There is no positive signal in the heart and pancreas throughout the hole experiment.To assessed various anatomic sites by FISH, the result shows that the positive signal mainly distributed in the macrophages in alveolar-septal and vascular endothelial cell, lymphoid nodule in the lymph node and the lymphocyte in the spleen. There are a few positive signals can be viewed in hepatic sinusoid、glomerulus and renal interstitium.In this paper, we establish the fluorescence in situ hybridization(FISH) and compared with the results of Immunofluorescence histochemical method and histopathological observation, find that the results of FISH paralleled results of Immunofluorescence histochemical method,which is also accordant with the histopathological observation.