Construction of a single-cycle replication recombinant infectious laryngotracheitis virus lacking the glycoprotein H gene and evaluation of its role in viral entry and infectivity

Infectious laryngotracheitis virus (ILTV), a significant avian pathogen belonging to the subfamily Alphaherpesvirinae, causes severe respiratory disease, particularly in unvaccinated flocks. In this study, we produced a recombinant ILTV lacking the essential envelope glycoprotein H (gH), a key determinant of viral entry and propagation. To achieve this, we engineered an ILTV mutant with the gH gene replaced by a BleCherry fluorescent reporter cassette. This modification enabled the identification and isolation of recombinant viruses through red fluorescence. To facilitate the replication of this gH-deficient mutant, we generated a stable Vero cell line expressing gH and a green fluorescent protein (GFP) reporter. This engineered cell line proved crucial for generating the recombinant ILTV and allowing controlled single-cycle replication. The recombinant ILTV exhibited enhanced viability across a range of multiplicities of infection (MOIs), although with a significant reduction in overall viral replication. Importantly, the modified virus is unable to replicate in the absence of exogenous gH, minimizing the risks associated with viral spread and unintended infections. This novel single-cycle recombinant ILTV platform holds significant promise for various applications, including safe gene delivery and the development of improved vaccine strategies for enhanced avian health management and disease control.