Identification Of A Heat-resistant Newcastle Disease Virus Strain HR09 And Construction Of Its Reverse Genetics System

Newcastle disease virus(NDV)is pathogen of the acute and highly infectious Newcastle disease(ND),which causes serious damage to the world poultry industry.The storage and transportation of heat resistant and attenuated NDV vaccines such as V4 and I-2 don't rely on cold chains,so they are widely used abroad,especially in hot climates and remote villages.In the 90s of last century,heat-resistant V4 strain was introduced in our country.However,because of the limitation of intellectual property rights,there is no commercial available in China.This study applied heat resistance test to the more than 40 NDV strains which were preserved in our lab previously and the high virulent and heat-resistant chicken-origin strain was screened(HR09 strain).The strain was determined the genome sequence and its molecular biological characteristics were analyzed.A reverse genetics system of HR09 heat resistant strain was established,then the nucleic acid sequences of F gene cleavage site was mutated,finally thermostable attenuated strain(NDV/rAHR09 strain)was obtained by reverse genetics system.1 Identification of NDV HR09 heat resistant strain and determination of its whole genome sequenceThe heat tolerance test was carried out with more than 40 chicken source isolates which were isolated and preserved by our lab,V4 strain and LaSota strain,and the hemagglutination(HA)activity after treated at 56 ? for 30min was used as the screening criteria,and a heat resistant HR09 strain was obtained.The biological characteristics were identified after plaque purification,results showed that the MDT is 57.6h,ICPI is 1.8,IVPI is 1.56,coincide with standard virulent strain characteristics.10 pairs of primers were designed and the whole genome sequence of HR09 strain were determined and bioinformatics were analyzed,results showed that the full-length genome sequence of the-strain is 15192nt,the amino acid sequence near the F protein cleavage site is 112RRQKRF117 and the length of the HN protein is 571,besides GC content is 46.27%.The similarity analysis of full length genome and amino acid sequences compared with heat-resistant V4 strain.I-2 strain is between 83.1%and 95.6%,and similarity analysis of six structural proteins about amino acid sequences is between 81.6%and 98.0%,phylogenetic tree based on F gene displayed HR09 strain is belonged to genotype VIII.2 Construction of reverse genetic operating system for heat resistant strain HR09Multiple primers were designed to construct full-length genomic cDNA clone of HR09 strain and three eukaryotic expression vectors containing P,NP and L gene separately.Then they were cotransfected into BSR-T7/5 cells and the virus strain NDV/rHR09 was successfully rescued.The result of rescued virus' heat resistance showed that the virus still had the HA activity after treated at 56 ? for 60min,indicating NDV/rHR09 strain and parental strain were similar.And MDT and ICPI were measured,while the MDT value is 60 h,ICPI value is 1.625,indicating NDV/rHR09 strain still with high virulence.3 Construction of thermostable attenuated strain NDV/rAHR09Two pairs of primers were designed for Overlap PCR method to mutant basic amino acids of F gene at the cleavage sites of 112,115 and 117 into non-basic amino acids with attenuated strain features.Then the transcription vector prNDV/HR09 was constructed and cotransfected into BSR-T7/5 cells with three auxiliary plasmids.The attenuated virus rNDV/AHR09 was successfully rescued.The strain still had the HA activity after treated at 56 ? for 45 min,and the MDT was longer than120h,and ICPI was 0.057,showed that the strain was attenuated.The results showed that the virulence of the rescued virus was still heat resistant and was significantly attenuated,and it was an ideal vaccine candidate.