Homologous Recombination Which Is An Important Mechanism Driving The Evolution Of NDV And Construction Of Full-length CDNA Of NDV Mukteswar Strain

As a serious avian disease of worldwide distribution, Newcastle disease cancause severe economic losses in the poultry industry since it was found in 1926. Thecausative agent of the disease, Newcastle disease virus (NDV), is a member of thegenus Rubulavirus of the subfamily Paramyxovirinae (family Paramyxoviridae, orderMononegavirales). NDV strains can be classified as highly virulent (velogenic),intermediate (mesogenic), or nonvirulent (lentogenic) on the basis of theirpathogenicity for chickens. Its negative strand RNA genome is approximate about15,100 nucleotides (nt) in size and contains six genes encoding the nucleocapsidprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F),hemagglutinin-neuraminidase (HN), and large polymerase protein (L). These genesare arranged in the order 3’-NP-P -M -F-HN-L-5’in its genome. In addition to thesegene products, additional proteins (desig-nated V and W protein) may be produced byan RNA-editing event that occurs during transcription of the P gene.NDV has high rates of mutation and replication and large population size.Therefore, it is notorious for its ability to evolve rapidly. Gene mutation at randomplays a key role during the course of evolution because RNA dependent RNApolymerase has not proofreading function. Homologous recombination is anotherimportant mechanism of biological evolution, which can result in more rapid changeof pathogen biology than gene random mutation. However, some researchers suggestthat genetic recombination is rare or does not occur in the case of negative-sense RNAviruses. Furthermore, although it has been reported that homologous recombinationcan occur in NDV by several groups, these mosaic recombinants are individualisolates. Therefore, there is no evidence supporting for the idea that recombinant virus is stable and can circulate in the field. In this study, a mosaic NDV lineage with mosicF gene is reported to reveal that recombinant viruses have genetic stability and canbecome prevailing strains. The mosaic NDV lineage includes D-JS-23-05,D-SD-21-05, D-SD-22-05, D-SD-24-05, D-ZJ-20-05, D-ZJ-26-05, D-ZJ-27-05,D-SD-29-05, D-SD-32-05, D-ZJ-28-05 and D-ZJ-33-05, and they were isolated fromlive domestic ducks in Eastern of China . Thus, homologous recombination is reallyan important mechanism driving the genetic diversity and rapid evolution of NDV. Ofcourse, it was required further experiments to verify, therefore, we are trying toconstruct a laboratory system to study homologous recombination between NDVs.To construct the lab system of homologous recombination in NDV, the reversegenetics system of NDV is necessary, which includes cloning of full-length cDNA ofsome NDV strains, cloning and expression of NP, P and L genes, construction of aBHK-21 cell line which could stably express T7 RNA polymerase and generation ofinfectious NDV from full-length cDNA. Due to the larger workload, only a full-lengthcDNA clone of NDV vaccine strain Mukteswar was constructed. It was assembledfrom subgenomic overlapping cDNA fragments which joined at shared restrictionsites, and ultimately, it was cloned in a low copy plasmid pMC18. Furthermore, itshould be noted that T7 RNA polymerase promoter was added at the beginning of thefull-length cDNA and the autocatalytic hepatitis delta virus ribozyme was added at theend.