The Function Of VP005L And VP092R Encoded By Infectious Spleen And Kidney Necrosis Virus(ISKNV)

Infectious spleen and kidney necrosis virus (Infectious Spleen and Kidney NecrosisVirus, ISKNV) is a typical species of the genus Megalocytivirus in the family iridoviridae,which causes a pandemic and serious disease in mandarin fish and severe damage tomandarin fish cultures in China. More and more studies have shown that mitochondriaplay a key role in the process of viral infection, and even play an important role in cellapoptosis after virus infection.We studied the funtion of VP005L and VP092R encoded by ISKNV and usedconfocal laser technology, subcellular localization technology and specific markerenzyme to confirm these two genes located in mitochondria.Here, JC-1reagent combined with flow cytometry to detect the change of themembrane potential transfected with VP005L.Compared with the control,experimental group had lower membrane potential. Annexin-V/PI reagents combinedwith flow cytometry, we detected the cells early apoptosis rate transfected with VP005L, compared with the control, experimental group’s early apoptosis rate wassignificantly higher than the control group. It proves that VP005L promote apoptosis.DsRNA technology combined with Annexin-V/PI,we detected the early apoptosisrate,the experimental group of early apoptosis rate was lower than the control group.Quantitative techniques with fluorescence detection after transfected with VP005L,apoptosis-associated gene expression were been detected. Compared with the control,VP005L had a little effect to the caspase3gene expression; and had downward effecton the Bcl-2gene, reduced by1.30times; it also upregulated Bax gene, up1.38times.These descriptions indicated VP005L participate Bcl-2regulation of mitochondrialpathway of apoptosis.Lu qing xia in our laboratory got the interactions between VP092R and mtHSP70protein.Based on this study, we discussed VP092R function further. B16cell lineswere screened by G418and then injected C57mice; we found that the experimentalgroup had higher tumor growth speed and bigger tumor. B16cell lines induced byactinomycin D (ACD), we use Annexin-V/PI reagents combined with flow cytometryto detected early apoptosis rate, the experimental group of early apoptosis rate waslower than the control group. DsRNA technology combined with Annexin-V/PI, wedetected the early apoptosis rate. The experimental group of early apoptosis rate washigher than the control group. Quantitative techniques with fluorescence detectionafter transfected with VP092R, apoptosis-associated gene expression were beendetected. Compared with the control, VP092R had a little effect to the caspase3geneexpression; and had downward effect on the Bcl-2gene, increased by3.43-fold; italso downregulated Bax gene by2.89fold. These descriptions indicated VP092Rparticipate Bcl-2regulation of mitochondrial pathway of apoptosis. Finally, afterinducedby ACD, we detected apoptosis-associated gene expression levels by westernblotting.Compared with the control group, the experimental group decreased P53, Bakprotein expression levels significantly,increased the expression of mtHSP70，but hadno change in caspase3and caspase9.From these results, we drawed VP092Rparticipate the endogenous apoptosis pathway with P53and mtHSP70.Through the study we found that, VP005Land VP092R are involved in mitochondrial apoptosis pathway and were regulated P53gene.It also laid thefoundation for exploring the mechanism of virus infection and host interactions.