The Cell Apoptosis Induced By Duck Reovirus In Bhk-21Cell

In this study, baby hamster kidney (BHK-21) were infected with five virus strains(JZ061214strain、HP080421strain、HP081122strain、HP09318strain and YM101120strain), which were isolated in our laboratory. The feature of pathological changes, the characteristics of gene structure, the proliferation of the virus, and apoptosis induced by duck reovirus were studied using cell culture technology, RT-PCR, light microscopy, real-time RT-PCR, electron microscopy, flow cytometry and TUNEL. Results of the study are as shown below:Pathological changes of BHK-21infected with four strains:The infection of monolayers of BHK-21with DRV caused a cytopathic effect(CPE) at24hour post infection(hpi). Infected cells were round and shrinked, the cytoplasm was filled with cavities, and some cells were dissolved. At48hpi, CPE was critical. Including multinucleated giant cells, a large number of died cells floating in the medium were observed. Infected cells died which were shown under light microscope by H.E, fragmented nucleus, pyknosis of nucleus and some was dissolved, multinuclear giant cells and acidophilic inclusion bodies were also seen. Ultrathin sections from infected cells were prepared, and the CPE was showed that there were some physalides without uniform size appeared in infected cells, which was monitored by electron microscopy.and virus particles with lattice arrangement were also observed, proliferation and replication of duck reovirus were in the cytoplasm, which were released by disintegration.Proliferation of DRV in BHK-21:the mixture of cells and cultural supertants were collected at1,3,6,12,24,48,72hpi. Viral content of every time interval was measured by means of RT-PCR.The results showed that1±12h post incubating HP080421、 YM101120and JZ061214, the titers were lower, but on the rise, so were HP09318, HP080421, and JZ061214at24～72hpi. Their contents peaks were reached at72phi.Apoptosis of DRV in BHK-21by flow cytometry:the number of HP09318and HP080421apoptotic cells reached the peak at3hpi, YM101120did it at6hpi, and JZ061214did it at12hpi. As the extension of inoculating DRV, the died cells number of later period was on the rise.Apoptosis of DRV in BHK-21making use of TUNEL:the number of apoptotic cells in DRV group at3hpi were more than those of control one. Apoptosis buffy nuclei could be observed. With the increase of died cells, the number of apoptosis was decreasing. This result was in line with detection by flow cytometry.Amplification of DRV S-class genes:the S2、S3and S3were amplificated, we acquired1251bp、1104bp and1104bp respectively, but the S1wasn’t acquired. The sequence results of phylogenetic trees indicated that the S2、S3and S4gene of the five isolated DRV strains were very close to DRV GZ/CHN/2007, they belong to the same branch, and the S2was also close to DRV NP03. Among five DRV strains, the relations of HP09318、HP081122、JZ061214and YM101120were nearest, YM101120and them belong to the same subline. The high homology of five DRV strains S2gene was98～100%. The homology of S2gene was about99%、97%and95%, respectively, compared the five isolated DRV strains with DRV GZ/CHN/2007、GRV03G and MDRV S12. The low homology of S2gene was77%～78%, compared the five isolated DRV strains with all ARV strains. The homology of the five isolated DRV strains S3gene reached up to98～100%. The homology of S3gene was99%.98%、97%and95%, respectively, compared the five DRV strains with DRV NP03、DRV091、DRV GZ/CHN/2007and MDRV03G. Their homology was also lower than ARV strains. The homology of five DRV strains S4gene was similar to those of S2gene.