Semi-quantification of antibody-dependent enhancement (ADE) in the uptake of Adenovirus serotype 5 into THP-1 cells.

Replication defective recombinant Ad5 vectors (rAdV5) are extensively explored for its applications in gene therapy and vaccine delivery. Ad5 enter into monocytes and macrophages through CAR independent route as an immune complex termed as antibody-dependent enhancement (ADE). We developed an effective method for estimating the ADE of rAdV5 encoding GFP (rAdV5-GFP) into THP-1 cells, using fluorimetric semi-quantification of GFP. Initially, twenty numbers of human sera samples were screened in HeLa cells for anti-Ad5 antibody titer using neutralization assay. Uptake of rAdV5-GFP in THP-1 cells was observed only after pre-incubation with the serially diluted human sera which are attributed to ADE. The optimal dilution which showed the maximum GFP expression as per the fluorescence microscopic analysis in THP-1 cells was used for further analysis. Fluorimetric analysis of the THP-1 cell lysate showed a maximum GFP intensity of 17058 RFU, which was equivalent to the 0.397 pmoles of Alexa Fluor 488 under the same experimental condition. Similarly, immunoblot analysis of GFP in THP-1 cell lysate and HeLa cell lysate confirmed the entry of rAdV5-GFP into the cells. The assay can serve as a platform for understanding the molecular events involved in ADE for the uptake of viruses into immune cells.

Mycophenolic acid (MPA) modulates host cellular autophagy progression in sub genomic dengue virus-2 replicon cells.

Cellular autophagy (Macrophagy) is a self-degradative process, executed through the network of autophagy associated genes (ATGs) encoded proteins. Both in vitro and in vivo studies suggest that dengue virus (DENV) induces autophagy and supports the viral genome replication and translation. Therefore, the cellular autophagy induced by dengue virus can be a good target for antiviral drug development. The action of mycophenolic acid (MPA), a specific inhibitor of DENV replication, was investigated in the stable BHK-21/DENV2 replicon cells. The inhibition was mediated by enhanced degradation of autophagic substrates in stable BHK-21/DENV2 replicon cells as evidenced by a decrease in lapidated LC3 (LC3II) and p62 expression in the presence of MPA. In contrast, the results indicated that four gene sets, namely Transmembrane protein 74 (TMEM74), Unc-51-like kinase 2 (ULK2), Cathepsin D (CTSD) and Estrogen receptor 1 (ESR1) were upregulated in stable BHK-21/DENV2 replicon cells, due to the sustained dynamic replication of DENV2 genome. These ATGs involved in the pre-autophagosomal structure (PAS) formation, were suppressed in the presence MPA. Instead, MPA induced the expression of different set of autophagy genes such as ATG4, AKT1, APP, ATG16L1, ATG16L2, B2M and HPRT1. An enzyme involved in the nucleotide salvage pathway, HPRT1, was highly expressed in the presence of MPA. The study shows that DENV2 replication is dependent on PAS formation and is inhibited in the presence of MPA by enhancing the degradation of autophagic substrates and suppression of PAS formation. This study provides impetus in designing MPA analogues to effectively inhibit dengue viral replication.