Production and Validation of Avian Influenza Pseudovirus

ABSTRACT

Avian influenza H5N1 remains a major health concern due to its pathogenicity and pandemic potential. Studying the virus directly requires high containment facility, which limits research accessibility. To facilitate this, this project aimed to develop a safe and suitable H5N1 pseudovirus system using HIV-1 derived lentiviral core, thus enabling the study of viral entry and immune responses under biosafety level 2 conditions. Glycoproteins hemagglutinin (HA) and neuraminidase (NA) genes from H5N1 that were commercially synthesised and cloned into the pTwist CMV BetaGlobin WPRE Neo vector and co-transfected into HEK293T cells alongside helper lentiviral plasmids encoding HIV Gag-Pol, Tat, Rev, and luciferase reporter for assessing infectivity of the pseudoviruses. The plasmids were verified by sequencing and restriction digestion. Western blot analysis detected the HIV capsid proteins within the cells as well as in the supernatant, indicating successful formation of lentiviral cores, while detection of HA confirmed its expression and incorporation into the pseudovirus. Hemagglutination assay further demonstrated receptor binding activity of HA. To summarize, this research project successfully produced and partially characterised the H5N1 strain of the influenza pseudoviral particles with the incorporation of functional HA. Despite the need for further optimisation of assays to detect glycoprotein and study infectivity, the system provides a safer and adaptable approach for studying entry mechanisms.