(A) Cell tropism of pCV-A6-luc. CV-A6 epidemiological surveillance. in the family in cells, it could also package EV-A71 and CV-A16 subgenomic RNA into infectious pseudoviruses and produced much higher titer of pseudoviruses. Thus pseudoviruses produced with CV-A6 capsid and CV-A16 subgenomic RNA were used for the following study, which designated as pCV-A6-luc. Open in a separate window Physique 1. Construction and verification of the single-round pCV-A6-luc contamination system. (A) Schematic map of enterovirus RNA genome and the pseudovirus encapsidation strategy of CV-A6. The CV-A6 capsid expressor was used to express all the structural capsid genes em in trans /em , the EGFP reporter gene was inserted at upstream of P1gene with a 2A protease self cleavage site (AITTL) and all other viral genome were deleted; Subgenomic replicons of CV-A6, EV-A71, CV-A16 and CV-B3 were constructed with the same strategy. Briefly, replicon was produced by replacing the capsid region with a firefly luciferase reporter gene and a 2A protease self cleavage site (KITTL) in the full length CV-A16 genome, and a T7 promoter was placed at the 5?end for transcription initiation. (B) Contamination of pCV-A6-luc would be neutralized by CV-A6 positive human plasma sample. 50 L of diluted pCV-A6-luc was mixed with an equal volume of serial diluted CV-A6 positive human plasma sample in duplicates. After incubation at 37C for 2?h, 100 L of RD cells (5??105/mL) was added and incubated at 37C in 5% CO2. Luciferase activity was measured at 16?h post infection. Nonlinear regression was performed with Graphpad Prism. (C) Specificity of pCV-A6-luc. 50 L of diluted pCV-A6-luc was mixed with an equal volume of serial diluted mouse anti-EV-A71, CV-A16, CV-A6, CV-B3 and CV-B5 serum samples in duplicates, respectively. After incubation at 37C for 2?h, 100 L of RD cells (5??105/mL) was added and incubated at 37C in 5% CO2. Luciferase activity was measured at 16?h post infection. As the capsid proteins of this chimeric pseudovirus came from CV-A6, pCV-A6-luc shared the comparable infectivity and Refametinib antigenicity with CV-A6 wild type computer virus. Contamination of pCV-A6-luc to susceptive cells could be inhibited by anti-CV-A6 NtAb positive human plasma sample in a dose dependent manner (Physique 1B), but could not be inhibited by mouse antisera raised against other Refametinib enteroviruses (EV-A71, CV-A16, CV-B3 and CV-B5) (Physique 1C). Taken together, we successfully built a strong pseudovirus packaging system to produce infectious CV-A6 pseudovirus with luciferase reporter. As pCV-A6-luc could only elicit a single round contamination and provided a superior surrogate for CV-A6 wild type computer virus, we next used it to develope a PVLA for anti-CV-A6 NtAb quantification. Optimization of the PVLA for anti-CV-A6 NtAb quantification We first compared relative infectivity of pCV-A6-luc on three common cell lines reported to be permissive to CV-A6 contamination. RD cell collection showed highest luciferase activity (relative light models, RLUs) upon pCV-A6-luc contamination, and thus it was chosen for further assay development (Physique 2A). Cell density was then optimized (Physique 2B). Although RD cells plated at a density of 1 1??105 cells per well gave a slightly lower RLUs value, the standard deviation (SD) was lower than that at a density of 1 1??106 cells per well (5.1% Vs 6.5%). Therefore, 1??105 cells per well was decided as the optimal input cell number. To enhance the incubation time, RD cells (1??105 cells per well) were infected with pCV-A6-luc (2??107 genome equivalents per well) in 96-well plates, luciferase activities Refametinib were measured after indicated incubation time (4C24?h). The results showed that RLUs increased over the incubation time and reached a plateau at 16?h (Physique 2C). Therefore, an incubation time of 16?h was determined for this PVLA. Further, pCV-A6-luc contamination system showed a good linear response between luciferase activity and the computer virus input over a broad range (4??105 to 4??107 genome equivalents per well) (r2? ?0.99) (Figure 2D). Open in a separate window Physique 2. Optimization of pCV-A6-luc PVLA. (A) Cell tropism of pCV-A6-luc. 2??104 cells were infected with 10 L of pCV-A6-luc (~?9??107 genome equivalents) and luciferase activity was measured at 16?h post infection. (B) Cell number optimization. 1 L of pCV-A6-luc (~?9??106 genome equivalents) was used to infect RD cells with various densities. Each density experienced 16 replicate wells. Luciferase activity was measured at 16?h post infection. (C) IL17RA Time course of pCV-A6-luc contamination. RD cells (1??105 cells per well) were incubated with 2 L pCV-A6-luc (~?2??107 genome equivalents) per well in 96-well plates, luciferase activity.
