Glycoprotein-Based Enzyme-Linked Immunosorbent Assays for Serodiagnosis of Infectious Laryngotracheitis

Abstract

Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes an acute respiratory disease in chickens known as infectious laryngotracheitis (ILT) that leads to significant economic losses to the poultry industry worldwide (1, 2). Currently, live attenuated vaccines are used to control ILTV infections. However, these vaccines are not satisfactory since the vaccine virus can revert to virulence after bird-to-bird passage (3) and can induce latent infections (4). Due to these limitations, live virus-vectored vaccines expressing the surface glycoproteins of ILTV have been developed as a safer alternative to attenuated live vaccines. A recombinant herpesvirus of turkey (HVT-LT) expressing ILTV glycoproteins D (gD) and I (gI) and Fowl pox virus (FPV-LT) expressing ILTV glycoprotein B (gB) and UL-34 genes are commercially used in the United States (5, 6). Recently, we have evaluated the role of ILTV gB, gC, and gD in immunogenicity and protection against a virulent ILTV challenge using recombinant Newcastle disease virus (rNDV) as a vaccine vector. Our results indicated that rNDV expressing ILTV gD provided complete protection against a virulent ILTV challenge in chickens (7). A vectored vaccine against ILTV infection will be safe without reversion of vaccine virus to be virulent or establishment of latency and also allow differentiation of vaccinated birds from the infected birds.The detection of the humoral immune response is critical for the rapid identification of ILTV-infected birds (5). The enzymelinked immunosorbent assay (ELISA) has been used for the detection of the humoral immune response. Despite its simplicity and rapidity, the commercially available ELISA using whole virus as an antigen is inefficient for detecting seroconversion with virus-vectored vaccines (6). Recently, individual ILTV surface glycoproteins have been used for ELISAs to detect ILTV antibodies in sera from vaccinated birds with attenuated and vectored vaccines against ILT (8-10). However, the specific glycoprotein-based ELISA has not been commercially available for rapid detection of seroconversion by vectored vaccines against ILTV. Therefore, in the present study, we developed rapid diagnostic ELISAs by using ILTV gB, gC, and gD (B-, C-, and D-ELISAs, respectively) as antigens, since these glycoproteins can induce humoral and cellmediated immune responses against ILTV and other herpesviruses (7,(11)(12)(13)(14). Each glycoprotein was eukaryotically expressed in insect cells. The diagnostic potential of these ELISAs was assayed with sera collected from chickens vaccinated with various virus-vectored vaccines. Furthermore, the efficacy of our ELISAs was validated by testing field serum samples and compared to that of a commercially available ELISA as a reference (fowl laryngotracheitis virus antibody test kit; Zoetis, San Diego, CA) (15-17).The ILTV gB, gC, and gD genes were cloned into the pCR 4 TOPO vector (Invitrogen) as described previously (7), and these genes were amplified from the TOPO vectors with concurrent introduction...