Development of a Novel Assay Based on Plant-Produced Infectious Bursal Disease Virus VP3 for the Differentiation of Infected From Vaccinated Animals.

Alessio Bortolami,Federica Gobbo,Selene Baschieri,Chiara Lico,Valentina Maria Panzarin,Andrea Fortin,Calogero Terregino,Francesco Bonfante,Carla Marusic,Elisa Mazzacan,Charifa Drissi Touzani,Marcello Donini

doi:10.3389/fpls.2021.786871

Abstract

Infectious bursal disease virus is the causative agent of Gumboro disease, a severe infection that affects young chickens and is associated with lymphoid depletion in the bursa of Fabricius. Traditional containment strategies are based either on inactivated or live-attenuated vaccines. These approaches have several limitations such as residual virulence or low efficacy in the presence of maternally derived antibodies (MDA) but, most importantly, the impossibility to detect the occurrence of natural infections in vaccinated flocks. Therefore, the development of novel vaccination strategies allowing the differentiation of infected from vaccinated animals (DIVA) is a priority. Recently, commercial vectored and experimental subunit vaccines based on VP2 have been proved effective in protecting from clinical disease and posed the basis for the development of novel DIVA strategies. In this study, an engineered version of the VP3 protein of IBDV (His-VP3) was produced in plants, successfully purified from Nicotiana benthamiana leaves, and used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-VP3 antibodies. The His-VP3 ELISA was validated with a panel of 180 reference sera and demonstrated to have 100% sensitivity (95% CI: 94.7–100.0) and 94.17% specificity (95% CI: 88.4–97.6). To evaluate the application of His-VP3 ELISA as a DIVA test, the novel assay was used to monitor, in combination with a commercial kit, detecting anti-VP2 antibodies, the immune response of chickens previously immunized with an inactivated IBDV vaccine, a recombinant Turkey herpes virus carrying the VP2 of IBDV (HVT-ND-IBD) or with plant-produced VP2 particles. The combined tests correctly identified the immune status of the vaccinated specific pathogen free white-leghorn chickens. Moreover, the His-VP3 ELISA correctly detected MDA against VP3 in commercial broiler chicks and showed that antibody titers fade with time, consistent with the natural decrease of maternally derived immunity. Finally, the novel assay, in combination with a VP2-specific ELISA, demonstrated its potential application as a DIVA test in chickens inoculated with VP2-based vaccines, being able to detect the seroconversion after challenge with a very virulent IBDV strain.

Highlights

Infectious bursal disease virus is the etiological agent of an acute, highly contagious viral disease that affects young chickens worldwide
The pBI-His-VP3 and the p35: AMCVP19 vectors were separately introduced in A. tumefaciens LBA4404 (Thermo Fisher Scientific, Rockford, IL, USA) and the two transformed strains were separately grown in the Luria Bertani medium overnight at 28◦C
N. benthamiana plants were infiltrated with a 1:1 mixed suspension of A. tumefaciens strains, carrying the pBI-His-VP3 or the p35:Artichoke Mottled Crinkle virus (AMCV)-P19 constructs (Figure 1A)

Summary

Introduction

Infectious bursal disease virus is the etiological agent of an acute, highly contagious viral disease that affects young chickens worldwide. Serotype 1 viruses can be further differentiated in classical and variant strains on the basis of their antigenic type (Mahgoub, 2012). Both classical and variant serotype 1 IBD viruses are pathogenic in chickens, causing mortality and immunosuppression, while serotype 2 viruses infecting both chickens and turkeys are nonpathogenic (Sharma et al, 2000). The worldwide distribution, the severe consequences of the infection, and the challenges in developing effective control strategies make IBD one of the most important diseases affecting commercial chickens (Van Den Berg, 2000; Eterradossi and Saif, 2019)

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