Résumé
Objective: Newcastle disease (ND) is the major viral infection of poultry inducing high morbidity, mortality, and significant economic impacts on the poultry industry. ND is caused by virulent strains of avian paramyxovirus serotype 1 (aPMV-1) which have the capacity to spread over long distances by animal and human movements. All strains of aPMV-1 belong to a single serotype and current vaccines have been used worldwide for a long time and demonstrated their efficacy in terms of clinical protection. Up to date vaccines have been made of old genotypes that emerged several decades ago. However, recent studies have shown that the virus has undergone significant evolution which has led to the progressive emergence of new genotypes with potential antigenic drifts. The recently described genotype XI in Madagascar contains original amino acid substitutions on F and HN proteins, some of them clustered in the head of the proteins, presumably exposed to the host immune system, suggesting that these substitutions may account for antigenic drifts. Indeed, we showed in vivo, under controlled conditions, that current vaccines conferred clinical protection against both the homologous genotype and genotype XI, but were unable to prevent virus shedding of heterologous genotype. Our objective was to produce genotype adapted vaccines, using reverse genetics to replace the F and HN of a live attenuated old-genotype virus (the genotype II Lasota strain) by the corresponding proteins of the more recent Madagascar genotype XI. This chimeric genotype XI-II will be characterized in vivo and evaluated in immunization/challenge trials. Methods: NDV minigenomes expressing eGFP under two promoters (pT7 polymerase and pCMV) have been constructed and compared in vitro. Subsequently, segments of the full genome of the genotype XI MG-725 strain and LaSota strain have been generated by reverse transcription, cloned and assembled under the control of CMV promoter. Rescue of the virus is done by co-transfection of the full length genome and plasmids expressing NP, P and L proteins in BHK21 cells. Results: The expression of the NP, P and L proteins of NDV MG725-08 strain has been confirmed on BHK 21 and by conventional RT- PCR on mRNA with specific NP, P and L primers. Then, the NDV rescue system has been validated using eGFP minigenomes in antisens position to demonstrate the function of the NDV ribonucleoprotein. These results have been confirmed by extraction of mRNA and RTqPCR or RT-PCR with specific eGFP primers. The NDV rescue system is now available to rescue the virus from an assembled full genome of NDV. Two genomes have been assembled from eight cloned segments and will be shortly rescued. Conclusion: Full length genome of NDV MG725_08 strain is now available and the NDV rescue system is available in vitro. The rescue of the LaSota strain will be achieved soon and the corresponding infectious clones characterized in vitro and in vivo on embryonnated SPF chicken eggs. The next step will be to replace the F and HN protein of the LaSota strain (live attenuated vaccine strain) by the corresponding proteins of the MG725-08 strain (the more recent genotype XI with a modification of the F protein to introduce an avirulent cleavage site) and to evaluate the protection and viral shedding in chicken experiments. (Résumé d'auteur)