Reverse Genetics Of A Prevalent Highly Virulent Genotype ⅦNDV Strain And Its Application

Newcastle disease (ND) is a highly contagious avian disease with a worldwide distribution. The causative agent of the disease, Newcastle disease virus (NDV), is an enveloped, nonsegmented single-stranded negative-sense RNA virus. NDV strains are classified into highly virulent, intermediate, and nonvirulent pathotypes on the basis of their pathogenicity in chickens. All NDV strains belong to a single serotype and can be divided into two classes. Class I and II viruses are further divided into different genotypes. Among these, genotype VII of the class II NDV strains has been associated with the most recent outbreaks in Asia, Europe, Africa, the Middle East, and South America. In China, genotype Ⅶd viruses predominantly circulating in the past decade have been responsible for most ND outbreaks in chickens.In a previous study, we genotypically characterized the NDV isolates recovered from chickens between2005and2012to establish the nature of the circulating genotype VⅦd strains in China. These Ⅶd NDV isolates were at least95.1%identical at the nucleotide level across the whole genome. However, they showed only82%-83%nucleotide sequence identity with vaccine strains such as La Sota, which are used as live vaccines against ND. The F and HN proteins of the isolates showed only87.4%-88.8%and86.7%-88.8%amino acid sequence identity with F and HN, respectively, of La Sota. The outbreaks of this disease in vaccinated poultry flocks in recent years are mainly attributed to the significant differences in the biology, serology, and genetics of the prevailing NDV strains and the current vaccine strains.NDV strain SG10is a highly virulent genotype Ⅶd NDV, which was isolated by our laboratory during an outbreak in a vaccinated broiler flock in2010. The F and HN proteins of the SG10strain share96.2%-99.8%and96.7%-99.7%amino acid sequence identity with those of the genotype Ⅶd NDV isolates recovered between2005and2012from chickens in China, respectively. In this study, a reverse genetics system for SG10was constructed. Using this system, we attenuated the velogenic SG10strain and assessed the potential utility of this attenuated virus as a vaccine and a viral vector. We first deleted6nt in UTR of SG10NP to construct the rNDV SG10-6bp. However, the newly generated mutated NDVs were not attenuated.After that, the cleavage site of virulent NDV SG10was modified to the consensus sequence of avirulent NDV. The newly generated mutated NDVs were highly attenuated. The construction of a reverse genetics system for NDV should allow the production of a vaccine vector for both vaccination and gene therapy. We inserted the EGFP/IB V S1genes between the P and M genes and successfully rescued the recombinant NDV aSG10-EGFP and aSG10-S1. EGFP or S1expression was observed in BSR T7/5cells infected with aSG10-EGFP or aSG10-S1. Therefore, the attenuated aSG10virus has potential utility as a viral vector.Efficacy studies in1-week-old chicks showed that strain aSG10more effectively protected the vaccinated birds from morbidity and mortality against highly virulent genotype Ⅶ NDV virus challenge compared to vaccine La Sota. Though the currently available vaccine La Sota could prevent disease but do not stop viral shedding, whereas aSG10not only prevent disease but also significantly reduce virus shedding, indicating aSG10is a promising vaccine candidate against NDV strains circulating in China.