Establishment Of A Reverse Genetics System For Newcastle Disease Virus VG/GA Strain And The Evaluation Of Its Optimal Insertion Site For Foreign Genes Expression

Newcastle disease virus (NDV) is the causative agent of Newcastle disease (ND),one of the most important poultry diseases worldwide, affecting a wide variety of birds andcausing significant economic losses to the poultry industry. NDV strains have been classifiedinto lentogenic, mesogenic or velogenic pathotypes. Velogenic strains are further classifiedinto viscerotropic velogenic and neurotropic velogenic strains. Viscerotropic velogenic strainsproduce lethal hemorrhagic lesions in the digestive tract with high mortality. Since the firstNDV strain was successfully rescued, several reverse genetics systems for different strains ofNDV have been generated. However, it is worthy of note that, most of the rescued NDVstrains are respirotropic strains, and the reverse genetics system of enteric NDV strains is stillunestablished. Therefore, the molecular biology and vaccinology development of entericNDV strains is lagging behind.The Villegas-Glisson/University of Georgia (VG/GA) vaccine strain of Newcastledisease virus (NDV) replicates both in the respiratory and intestinal tract, with a preferentialtropism for the latter. After vaccination with the VG/GA strain, solid immunity to virulentNDV is conferred at both the upper respiratory tract and intestinal tract as evidenced byresistance to virulent NDV challenge. Thus, the VG/GA strain has become a routinely usedlive vaccine to prevent Newcastle disease throughout the world, especially against velogenicviscerotropic NDV.In the present study, aimed to develop an infectious clone of enteric NDV, wechoose the NDV VG/GA vaccine strain as parental virus to generate an enterotropic vaccinevector. Firstly, a full-length cDNA clone (FLC) of the VG/GA strain was constructed byassembling four RT-PCR fragments of the virus into a transcription vector. Then, an infectiousvirus rVG/GA was rescued by co-transfecting the FLC and supporting plasmids expressingthe NP, P, and L proteins of NDV into MVA-T7infected HEp-2cells, and confirmed by HAtest, HI test and whole genome sequencing. Besides, in vitro characterizations, such as EID50,TCID50, MDT, ICPI and growth curve showed that the rescued rVG/GA virus not only remained its lentogenic pathotype, but also kept its reproduction characteristics and growthability.For better understanding and systematically evaluating the optimal insertion siteforNDV VG/GA strain foreign gene expression, the green fluorescence protein (GFP) openreading frame was inserted into different intergenic (IG) regions of the VG/GA strain viralgenome in the full-length cDNA clone. The recombinant viruses vectoring the GFP at thedifferent insertion sites (NP/P, P/M, M/F, F/HN and HN/L) were rescued using reversegenetics technology. In vitro and in vivo characterizations, such as EID50, TCID50, MDT andICPI showed that the rescued viruses rVG/GA-GFP-NP/P、 rVG/GA-GFP-P/M、rVG/GA-GFP-M/F、rVG/GA-GFP-F/HN and rVG/GA-GFP-HN/L retained their lentogenicpathotype and no virulence reversion was demonstrated. In the meanwhile, the result of thegrowth curves showed that the all of the rescued viruses kept their reproductioncharacteristics, and displayed slightly delayed growth kinetics due to the insertion of anadditional gene. This effect was most prominent when the GFP gene was inserted at the IGregions closer to the3’ end of the viral genome. Quantification of GFP mRNA expressionby real-time RT-PCR suggested that there is a gradient of mRNA abundance according to thegene position. In addition, the measurement of GFP fluorescence intensity from the infectedDF-1cells showed that the recombinant virus with GFP insertion at the P/M site had thehighest GFP expression. Overall results suggest the noncoding region between the P and Mgenes is the optimalinsertion site for foreign genes expression by the VG/GA NDV vaccinevector.