| [Objectives]To construct an expressing plasmid of varicella-zoster virus (VZV) ORF62 as a standard control plasmid for real-time fluorescence quantitative PCR, and to establish a novel assay for quantifying cell-associated virus (CAV) with a reporter cell line for VZV, MV9G cells, with an aim to screen for anti-VZV compounds and to investigate its antiviral mechanisms.[Methods]1. Complete ORF62 sequences of VZV Oka vaccine strain purchased from Changchun BCHT Biotechnology Corporation (BKvOka) was amplified by PCR, and then cloned into a highly efficient expressing plasmid pCAGGS, to construct a BKvOka ORF62-expressing plasmid. Double enzyme digestion and DNA sequencing were used to verify the inserted ORF62 fragment.2. MV9G cells were co-cultured with serially diluted BKvOka-infected MeWo cells containing CAV for 72 hours. The luciferase activities of MV9G cells activated by BKvOka CAV were tested with chemiluminescence and shown as relative light unit (RLU). The VZV foci formed on MV9G monolayer were stained by indirect immunoperoxidase method with a monoclonal anti-VZV IE62 antibody and counted under microscope. The VZV DNA copies were detected by SYBR Green real-time PCR with a primer set targeting VZV ORF62. The ratios of RLU/ foci and RLU/ DNA copies were calculated and their correlations were analyzed. Standard curves of RLU-foci, RLU-DNA copies, and DNA copies-foci were then established. Acyclovir (ACV) was added in the medium to inhibit viral growth, and the standard curves of RLU-foci, RLU-DNA copies, and DNA copies-foci in the presence of ACV were established with the methods above.3. MV9G cells were co-cultured with randomly diluted VZV Oka vaccine strain purchased from GlaxoSmithKline Corporation (GSKvOka) containing CAV for 72 hours. Luciferase activities (RLU) of MV9G cells and viral foci and DNA copies of GSKvOka were tested by the methods above. Expected DNA copies and focus numbers were calculated according to the ratios from the standard curves in the absence of ACV. And then, the expected values were compared with the tested values to validate the applicability of reporter cell line MV9G in quantifying CAV.[Results]1. The inserted ORF62 fragment was verified by both double enzyme digestion and DNA sequencing. A recombinant ORF62-expressing plasmid pCAG-BKvOka62 was thus successfully constructed.2. After 72-hour co-culture of MV9G cells with serially diluted BKvOka CAV, the luciferase activities (RLU) activated by BKvOka CAV were statistically correlated with both focus numbers (r = 0.999, P<0.05) and DNA copies (r = 0.974, P < 0.01). DNA copies were correlated with VZV focus numbers as well (r = 0.979, P < 0.01). The ratios of RLU/ foci and RLU/ DNA copies were 181.62 and 1.97×10-4, respectively. In the presence of ACV, the luciferase activities (RLU) activated by BKvOka CAV were statistically correlated with both focus numbers (r = 0.991, P < 0.01) and DNA copies (r = 0.971, P < 0.01), DNA copies were correlated with focus numbers, too (r = 0.936, P < 0.05).3. After 72-hour co-culture of MV9G cells with randomly diluted GSKvOka CAV, the luciferase activities (RLU) activated by BKvOka CAV were statistically correlated with both focus numbers (r = 0.992, P < 0.01) and DNA copies (r = 0.988, P < 0.01), DNA copies were correlated with VZV focus numbers as well (r = 0.966, P < 0.01). The expected DNA copies and focus numbers based on luciferase activities (RLU) were basically matched the tested values.[Conclusions]1. A BKvOka ORF62-expressing plasmid pCAG-BKvOka62 was constructed successfully, which provided a standard control plasmid for real-time fluorescence quantitative PCR test for VZV DNA copies.2. In co-culture of MV9G cells with known amount of BKvOka CAV, the luciferase activities (RLU) of MV9G cells activated by BKvOka CAV were correlated with both focus numbers and DNA copies in the presence or absence of antiviral compounds ACV. In co-culture of MV9G cells with unknown amount of GSKvOka CAV, the luciferase activities (RLU) of MV9G cells activated by GSKvOka CAV were correlated with both focus numbers and DNA copies, too. The expected focus numbers and DNA copies of GSKvOka CAV based upon luciferase activities (RLU) activated by GSKvOka CAV were basicly consistent with its tested values. The luciferase activity (RLU) changes of MV9G cells activated by CAV co-culture definitely revealed changes of viral focus numbers and DNA copies, MV9G cells were thus applicable to quantify CAV.3. For quantifying CAV, immunoperoxidase staining of viral foci and real-time PCR test of DNA copies were laborious, subjective, or easy to be contaminated, the VZV reporter cell-based method was simple, rapid, sensitive, and objective, which provided a high-throughput assay for screening anti-VZV compounds and investigating its antiviral mechanism.
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