Genetic predisposition of human viral influenza A (H1N1- PR8) in cancer cell lines

Abstract

Background: Research has made significant progress, with evidence indicating that enzymes that deplete amino acids may reduce the risk of various cancers. Influenza viral infections exhibit a wide host range, including birds, humans, and various other well-adapted species, and can evolve into highly pathogenic strains. The scientists sought to identify methods to mitigate this invasion, which poses a significant threat to human survival. Subjects & methods: This study focuses on the purification of L-glutaminase, a medically significant enzyme, from Bacillus subtilis. The purification process involved 70% ammonium sulfate precipitation, followed by diethylaminoethyl cellulose (DEAE-C) ion exchange chromatography and gel filtration chromatography using Sephadex G-200. From the stock solution, various concentrations (12.5, 25, and 50 μg/ml) of L-glutaminase were derived to assess their efficacy against human influenza A/Puerto Rico/8 H1N1 (IAV/PR8). The objective was to evaluate the impact of L-glutaminase on viral replication in MDCKII and A549 cells, employing plaque assays and reverse transcriptase quantitative real-time polymerase chain reaction to analyze the gene expression of specific viral proteins, including non-structural protein 1 (NS1) and nucleoprotein (NP). Results: The elution profile for L-glutaminase on the DEAE-C column revealed that the most active fractions (40-50) exhibited an enzyme activity of 200 U/mg protein and a purification fold of 21. The enzyme's specific activity at this stage was 12.5 U/mg, yielding a purification fold of approximately 8.3 and a 50% recovery of the enzyme. Results: Our findings indicated a significant decrease in the ability of IAV/PR8 to grow and replicate in MDCKII cells at multiplicities of infection (MOI) of both 0.1 and 0.01. At MOI of 0.01, plaque-forming units (PFU) per milliliter significantly decreased at concentrations of 25 and 50 μg/ml after 24 hours. Similarly, the expression of NP and NS1 proteins of IAV/PR8 was markedly downregulated at concentrations of 25 and 50 μg/ml but 8 hours. In conclusion, L-glutaminase demonstrated antiviral efficacy against the replication of IAV/PR8, likely due to its influence on the production of viral proteins NP and NS1, which are crucial for viral protein synthesis.