The Reseach Of Six Nucleotides In The5’ Untranslated Region Of The NP Gene Affect The Virulence Of Goose Newcastle Disease Virus NA-1Strian Using Reverse Genetic System

Newcastle disease (ND) caused by Newcastle disease virus (NDV) is acontagious disease affecting wild birds and domestic poultry that frequently resultsin large economic losses. Highly pathogenic ND is a rapidly spreading disease ofpoultry, with four worldwide pandemics since its emergence in the early20thcentury.The most recent pandemic was caused by genotypes VII and VIII in late1980s and1990s. For decades, this virus was not considered to be infectious for mammals andwaterfowl. However, cases of waterfowl infection with NDVs have become frequentin China since the late1990s. The virulent goose genotype VII NDV strain NA-1was isolated from an epizootic in flocks of geese in Jilin province, NortheasternChina. According to recent reports, virulent strains isolated from geese wereresponsible for outbreaks of ND in waterfowl and belonged to genotype VII.Virulent NDV strains are still isolated frequently in vaccinated birds,demonstrating that NDV remains a sustained threat to commercial flocks. Nowadays,the prevailing NDV strain of geese in China is genotype VII, and therefore vaccinestrains such as B1, Clone30, La Sota, and V4are used to protect geese from NDVinfection. Compared with prevailing NDV strains, these vaccine strains belong todifferent genotypes from NA-1. The antigenic differences between the prevailing andvaccine strains might explain current ND outbreaks in vaccinated poultry flocks.In order to prevent and control this disease, the homologous vaccine isnecessary. The amino acid sequence at the F protein cleavage site has been shown tobe a major determinant of NDV virulence although other genes also have effect onNDV virulence. Transcriptional and translation control signals may also modulate virulence by controlling protein expression as has, for instance, been described forcanine distemper virus and NDV. NA-1has6additional nts inserted in the5′untranslated region (UTR) of the NP gene between1,648and1,653nts whencompared with the La Sota strain. The6additional nts may affect the virulence.In this study, NA-1strain is the parent virus, rNA-1(-) strain has asix-nucleotide deletion in the5′untranslated region (UTR) of the NP gene. First ofall, six pairs of specific primers were designed to amplify the complete genomiccDNA. PCR products named A, B, C, D, E and F were purified and cloned intopMD18-T vector. A full-length cDNA of NA-1was constructed using a modifiedpBR322clone vector, designated pAF.Subsequently, using the plasmids constructed with segment A and the pMD18-Tvector (named T-A), six additional nucleotides between1648and1653nts in the5′UTR of the NP gene were deleted using PCR. Plasmids were transformed intocompetent cells and clones which contained the six nucleotide deletion6[namedT-A(-)] were screened via PCR. To construct a full-length cDNA clone which has asix-nucleotide deletion in the5′untranslated region (UTR) of the NP, T-A(-) wasused to replace the same segment in pAF using SalI and AgeI, designated pAF(-).Finaly, Infectious viral particles were obtained after A full-length infectiouscDNA clone and helper plasmids were co-transfected into BHK-21cells expressingT7RNA polymerase. The biological characterization of the recombinant virusshowed that the rNA-1(-) virus had similar growth ability to its parental NA-1inDF-1cells. However, the pathogenicity of this mutant virus in embryonating chickeneggs and day-old chickens decreased, as evidenced by a longer mean death time andlower intracerbral pathogenicity index when compared with the parental virus. Theresults suggest that the6nts from1648nts to1653nts in NP gene UTR of NDVNA-1strain may contribute to the reduced virulence in some genotype VII NDV.Controlling and preventing ND have great epidemiological significance. Thebest method to solve this problem is the development of homologous vaccines withhigh efficiency and safety. However, no current commercial vaccine is available for geese against virulent genotype VII NDV. Therefore, we set out to establish a reversegenetics system that allows the genetic manipulation of virulent strain, which couldbe used as a vaccine candidate or a viral vector to express foreign genes in ourongoing studies.