Q-PCR Analysis Of BmNPV Nucleic Acid Replication And Gene Expression Inhibited By Prodigiosin

Bombyx mori nuclear polyhydrosis virus(Bm NPV) belongs to the baculoviridae family of rod-shaped enveloped viruses that containing a circular double-stranded supercoiled DNA genome, which possess two different phenotypic viral particles during the post infection process, the budded virus(BV) and the occlusion-derived virus(ODV). BV spread infection among cells which mediated by membrane fusion proteins GP64, while ODV formed in the late phase of viral post infection, embedded in the stable protein crystals, which serve to spread infection among individuals. The gene expression of Bm NPV can be generally divided into early and late categories, which may be further divided into immediate early, early, late and very late phase, Previous studies have proved that several viral genes, including ie-1, vp39 and p10 could be used as genetic markers to evaluate viral immediate early, late and very late phases, respectively.Prodigiosin, a bacterial secondary metabolite with three tripyrrole rings, exhibits various biological activities, our previous study found that prodigiosin shown a strong inhibitory activity against Bm NPV. Therefore, the aim of this study was to further investigate the bioactivitiy against Bm NPV. The main contents and results are as follows: 1. The influence of prodigiosin on the production of budded virus(BV) and the formation of OB in Bm NS cellsThe virus-containing supernatants were harvested at 72 h post infection,plaque assay and plaque forming units were applied to evaluate BV tiers. The results show that prodigiosin can inhibit the production of BV effectively, the BV tiers in non-prodigiosin control group are 105 and 103 fold higher than 100 n M and 10 n M prodigiosin-treated cells. The OB-producing cells were counted and recorded by using inverted microscope, and the results showed that prodigiosin significantly inhibited the formation of OB in Bm NS cells. With the increase of prodigiosin concentration, the number of OB-producing cells was gradually reduced which down to the bottom when the prodigiosin concentration was 100 n M. 2. The influence of prodigiosin on viral DNA replicationThe q-PCR technology was applied to analyze the replication of viral DNA. Unlike the control groups which showed significant increase of viral DNA, the viral DNA abundance in prodigiosin-treated cells was much slower. Besides, the viral DNA abundance in control groups was much higher than prodigiosin-treated cells. At 48 h, 72 h and 96 h post infection, the amount of viral DNA in prodigiosin-treated groups was as low as 3.0, 1.3 and 5.7% of that in DMSO-treated cells(control), respectively. All these results indicated that prodigiosin significantly inhibited the viral DNA replication. 3. The influence of prodigiosin on Bm NPV gene transcriptionThe resault of q RT-PCR analysis indicated that the transcription levels of all tested genes(ie-1, vp39, p10)were significantly inhibited by prodigiosin. At 72 h post infection, the expression level of viral ie-1 gene in DMSO-treated cells was 55 fold higher than that of in prodigiosin-treated cells. The expression level of viral vp39 gene in prodigiosin-treated cells was as low as 1.1 % of that in DMSO-treated cells. The expression of viral p10 gene in prodigiosin-treated cells was extremely low that barely detected. 4. The inhibitory effect of prodigiosin on Bm NPV mediated cell membrane fusionWe found that prodigiosin inhibited membrane fusion of Bm NPV-infected cells under low p H, Bm NPV-infected cells treated with prodigiosin or DMSO were incubated in low p H TC100 medium for 5min. Lots of syncytiums were obeserved in DMSO-treated cells, whereas no syncytium was detected in prodigiosin-treated cells. The results indicated that Bm NPV mediated cell membrane fusion was significantly blocked by prodigiosin treatment.