Identification and functional analysis of membrane proteins gD, gE, gI, and pUS9 of Infectious laryngotracheitis virus.

Herpesvirus envelope proteins are of particular interest for development of attenuated live, marker, and subunit vaccines, as well as development of diagnostic tools. The unique short genome region of the chicken pathogen infectious laryngotracheitis virus (ILTV, Gallid herpesvirus 1) contains a cluster of six conserved alphaherpesvirus genes encoding membrane proteins, of which up to now only glycoproteins gG and gJ have been analyzed in detail. We have now prepared monospecific rabbit antisera against ILTV gD, gE, and gI, and the ILTV type II membrane protein pUS9, each of which showed specific immunofluorescence reactions, and detected proteins of approximately 65 and 70 kDa (gD), 62 kDa (gI), 75 kDa (gE), or 37 kDa (pUS9) in western blot analyses of infected chicken cells. The proteins gD, gI, and gE, but not pUS9, were identified as abundant virion proteins, and gE and gI were shown to be N-glycosylated. We also isolated gE-, gI-, and pUS9-deleted ILTV recombinants, whereas it was not possible to purify gD-negative ILTV to homogeneity, indicating that gD, like in other alphaherpesviruses, is essential for receptor binding and virus entry. The pUS9-deleted ILTV exhibited almost wild-type-like replication properties in cell culture. The gE- and gI-negative viruses showed significantly reduced plaque sizes, whereas virus titers were barely affected. Since homologous gene-deletion mutants of other alphaherpesviruses are in use as live vaccines, the generated ILTV recombinants might be also suitable for this application.