Potency of bacterial sialidase Clostridium perfringens as antiviral of Newcastle disease infections using embryonated chicken egg in ovo model

Abstract

Background and Aim: Clostridium toxins are widely used as medicinal agents. Many active metabolic enzymes, including sialidase (neuraminidase), hyaluronidase, and collagenase, contribute to the mechanism of action of these toxins. Sialidase from Clostridium perfringens recognizes and degrades sialic acid receptors in the host cell glycoprotein, glycolipid, and polysaccharide complexes. Sialic acid promotes the adhesion of various pathogens, including viruses, under pathological conditions. This study aimed to investigate the potential of C. perfringens sialidase protein to inhibit Newcastle disease virus (NDV) infection in ovo model. Materials and Methods: C. perfringens was characterized by molecular identification through polymerase chain reaction (PCR) and is cultured in a broth medium to produce sialidase. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis was conducted to characterize the sialidase protein. In contrast, enzymatic activity and protein concentration were carried out using a neuraminidase assay kit and Bradford to obtain suitable active substances. Furthermore, embryonated chicken egg models were used to observe the toxicity of several sialidase doses. Then, the hemagglutination (HA) titer was obtained, and absolute quantitative reverse transcription–PCR assay was performed to measure the viral replication inhibitory activity of sialidase against NDV. Results: Each isolate had a specific sialidase gene and its product. The sialidase derived from C. perfringens could hydrolyze the sialic acid receptor Neu5Ac (2,6)-Gal higher than Neu5Ac (2,3)Gal in chicken erythrocytes, as observed by enzyme-linked lectin assay. A significant difference (p = 0.05) in the HA titer in the pre-challenge administration group at dosages of 375 mU, 187.5 mU, and 93.75 mU in the competitive inhibition experiment suggests that sialidase inhibits NDV reproduction. Quantification of infective viral copy confirmed the interference of viral replication in the pre-challenge administration group, with a significant difference (p = 0.05) at the treatment doses of 750 mU, 375 mU, and 46.87 mU. Conclusion: The potency of sialidase obtained from C. perfringens was shown in this study, given its ability to reduce the viral titer and copy number in allantoic fluids without adversely impacting the toxicity of the chicken embryo at different concentrations.

Clostridium perfringens Sialidase Interaction with Neu5Ac α-Gal Sialic Acid Receptors by In- Silico Observation and Its Impact on Monolayers Cellular Behaviour Structure

“…In previous studies (evaluation and potency) sialidase was known to have the potential to inhibit viral infection both in ovo and in vitro, this was supported by a decrease in the ability of viral replication in exposed host cells. 11,13,24 This study assure and encourage this discovery as shown by the ability of sialidase which has the lowest Gibbs energy value (ΔG) − 7.35 with a Ki value of 4.11 µM towards sialic acid α (2.3)-Gal receptors. This demonstrates its capacity to outcompete viral molecule competitors and attach to sialic acid receptors.…”

“…Puri ed sialidase enzyme activity was observed using the Neuraminidase assay kit MAK121 (Sigma-Aldrich) with appropriate protocol procedures to obtain a quantitative value in U/ml. 13,14 Chicken Embryo Fibroblast Cell Culture Treatment By Sialidase CEF monolayer cultured cells were obtained from SPF embryonated chicken eggs (ValoBioMedia GmbH, German) age of 8-10 days. Embryo was taken and rinsed brie y with Phosphate Buffered Saline (PBS).…”

“…Bacterial sialidase can catalyze the hydrolysis of terminal sialic acid linked by α (2,3) -, α (2,6) -, or α (2,8) bonds to a wide variety of substrates. [11][12][13][14] In addition, some of these enzymes can catalyze the transfer of sialic acid from sialoglycans to asialoglycoconjugates by means of the transglycosylation reaction mechanism. 15 Research regarding to the inhibition of viral infection by sialidase has been carried out in vitro using cell culture and in vivo by animal models.…”

Clostridium perfringens sialidase interaction with Neu5Ac α-Gal sialic acid receptors by in-silico observation and its impact on monolayers cellular behavior structure

“…The interaction between sialidase NanI and NanJ with sialic acid receptors has been successfully illustrated, predicted, and analyzed in this study using in-silico analysis of a 3D model of the sialidase enzyme from C. perfringens obtained from the PDB data bank. The potency and evaluation of sialidase were known to have the ability to inhibit viral infection both in ovo and in vitro ; this was supported by a decrease in the ability of viral replication in exposed host cells [11– 13] . This study assured and encouraged this discovery, as shown by the ability of sialidase, which has the lowest Gibbs energy value (ΔG) –7.35 with a Ki value of 4.11 µM, towards sialic acid α(2.3)-Gal receptors.…”

“…Afterward, the dialyzed residue was purified by ion exchange using Q Sepharose ® Fast Flow (Merck, Germany), and final purification was performed using affinity chromatography with oxamic acid agarose before being kept at −20°C. Purified sialidase enzyme activity was observed using the Neuraminidase assay kit MAK121 (Sigma-Aldrich) with appropriate protocol procedures to obtain a quantitative value in U/ml [13 , 14] .…”