Azadmanesh, K. | Search bioRxiv

bioRxiv2023

The chicken chorioallantoic membrane model for isolation of CRISPR/cas9-based HSV-1 mutant expressing tumor suppressor p53

Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, which selectively target and kill cancer cells while sparing normal ones. Among them, engineered Herpes Simplex Virus type 1 has been proposed to be employed as a potential treatment of cancer and was moved to phase III clinical trials. In this study, to improve oncoselectivity and oncotoxicity properties, the UL39 gene of the ICP34.5 deleted HSV-1 was manipulated with the insertion of the EGFP-p53 expression cassette utilizing CRISPR/ Cas9-mediated editing genome. The {Delta}UL39/{Delta}{gamma}34.5/HSV1-p53 mutant was isolated using the chorioallantoic membrane (CAM) of fertilized chicken eggs as a complementing membrane to support the growth of the viruses with gene deficiencies. Phenotypic characterization of {Delta}UL39/{Delta}{gamma}34.5/HSV1-p53-infected cells was compared with the parent {Delta}{gamma}34.5/HSV-1 in vitro. Our results indicate that the CAM model can be a promising strategy for isolating recombinant virus such as HSV-1-P53 that is unable to replicate in cell lines due to the death induced by exogenous p53 during virus replication.

microbiology↗

bioRxiv2020

Optimizing Consensus Generation Algorithms for Highly Variable Amino Acid Sequence Clusters

Producing a functional consensus sequence is a preliminary bioinformatics task, which is a necessity for many research purposes. However, the existence of hypervariable regions in the input multiple sequence alignment files causes complications in generating a useful consensus sequence. The current methods for consensus generation, Threshold, and majority algorithms, have several problems, which exclude them as applicable algorithms for such highly variable sequence clusters. Hence, we designed a novel alternative algorithm for the same purpose. The algorithm was explained both using a mathematical formula and a practical implementation in Python programming language. A sequence set from HCV genotype 1b E2 protein has been utilized as a practical example for evaluating the algorithms performance. A few in silico tests have been performed on the output sequence and the results have been compared to results from other algorithms. Epitope-mapping analysis indicates the functionality of this algorithm, by preserving the hotspot residues in the consensus sequence, and the antigenicity index shows significant antigenicity of the consensus sequence. Moreover, phylogenetic analysis shows no significant change in the placement of the new consensus sequence on the phylogenetic tree compared to other algorithms. This approach will have several implications in designing a new vaccine for highly variable viruses such as HIV-1, Influenza, and Hepatitis C Viruses (HCV).

bioinformatics↗