Exploration Of The Novel Heat-resistant Newcastle Disease Virus Expressing GB?gD And GI Proteins Of Infectious Laryngotracheitis Virus

Infectious laryngotracheitis(ILT)in chicken was found to be caused by Infectious laryngotracheitis virus(ILTV)caused by the characteristics of dyspnea,asthma,coughing up bloody mucus and decreased egg production,poultry production has brought great economic losses.The construction of genetically engineered recombinant vaccine using Newcastle disease virus(NDV)as vector is one of the important methods to prevent ILT in production,but the traditional NDV attenuated strain has disadvantages such as heat resistance,preservation and transportation.Therefore,it is of great significance to construct a novel heatresistant NDV recombinant vector vaccine strain.A heat-resistant and virulent NDV strain NDV/HR09 was isolated and identified in our laboratory earlier,and a novel heat-stable chimeric virus NDV/cHR-DT-CI was constructed on this basis,whose F gene was replaced by the F gene of genotype ? strain.The cleavage site of F protein was mutated to 112ERQER ? L117(NDV ClassI).In this study,three recombinant vaccine candidate strains expressing ILTV gB,gD and gI proteins were constructed by reverse genetic technology using this heat-resistant attenuated NDV strain as the mother virus,and their biological characteristics were identified.This study provides an important technical basis for the development of ILT vaccine.1.Construction of a heat-resistant Newcastle disease virus exogenous gene expression vectorIn this study,the NDV/cHR-DT-CI reverse genetic platform was used to analyze the whole genome of the virus.The Pme I site was introduced into the non-coding region between P and M genes of NDV genome,and the pNDV/rAHR09-?-PI containing the Pme I site was constructed.The constructed transcription vector was co-transfected with three helper plasmids(pCI-NP,pCI-P and pCI-L)into BSR cells for virus rescue and identification.The results showed that the hemagglutination of allantoic fluid of SPF chicken embryo inoculated with transfected cells and supernatant was positive,indicating that the expression vector pNDV/rAHR09-?-PI containing Pme I site was successfully constructed.To verify whether the vector pNDV/rAHR09-?-PI can be used for the expression of foreign genes,In this study,Enhanced Green Flurescent Protion(EGFP)gene was recombinant into the Pme I site of ND V expression vector pNDV/rAHR09-?-PI by seamless cloning.The transcription vector pNDV/rAHR09-?-EGFP containing EGFP gene and whole NDV gene was constructed.After the virus was saved,green fluorescence could be observed under fluorescence inverted microscope 48 h after transfection,indicating that the recombinant virus was successfully constructed.The saved recombinant virus was named pNDV/rAHR09-?-EGFP.In order to investigate the biological characteristics of the recombinant virus,the recombinant virus pNDV/rAHR09-?-EGFP was continuously transmitted to chicken embryos for 20 generations,and the EGFP gene in different generations was amplified by PCR.The results showed that EGFP gene could be identified in each generation of recombinant virus,and the sequencing results were correct,indicating that the recombinant virus had good genetic stability.EGFP in the recombinant virus was identified by Western Blot.The results showed that the recombinant virus could express EGFP protein normally.Then,in order to investigate whether the insertion of exogenous genes would affect the heat resistance of the recombinant virus,the recombinant virus was treated at 56? for 10 min,20 min,30 min,40 min,50 min and 60 min,respectively.The results of hemagglutination showed that the hemagglutination titer of the recombinant virus under heat treatment remained at 8log2,which proved that the recombinant virus had good thermal stability.Finally,this study evaluated the infectivity and virulence of the recombinant virus to chicken embryos.The results showed that the infectivity of the recombinant virus to chicken embryos was 108EID50/0.2 mL,and the MDT value was 148 h,which was consistent with the characteristics of NDV attenuated strain,indicating that exogenous EGFP gene had no effect on the virulence and pathogenicity of NDV.The above results indicate that the present study successfully constructed an NDV expression vector inserted at the Pme ? site,which can successfully express the exogenous EGFP gene.Meanwhile,the heat resistance and virulence of recombinant virus were not affected by the insertion of exogenous gene.2.Construction of recombinant heat-resistant Newcastle disease virus expressing gB?gD and gI proteins of ILTVBased on the constructed expression vector pNDV/rAHR09-?-PI,gB,gD and gI genes of ILTV were inserted into the Pme ? site,respectively,to construct heat-resistant recombinant Newcastle disease virus expressing ILTV gB,gD and gI proteins.The three strains were named NDV/rAHR09-?/ILTV-gB,NDV/rAHR09-?/ILTV-gD and NDV/rAHR09?/ILTV-gI.The polyclonal antibodies against gB,gD and gI proteins were used as primary antibodies,and the expression of exogenous proteins of the 3 recombinant viruses was identified by Western Blot.The results showed that all the 3 recombinant viruses could express exogenous proteins.In order to evaluate the genetic stability of the three strains of recombinant virus,the three strains were transmitted continuously for 20 generations on SPF chicken embryos,and the allallic fluid of the virus at generations 0,5,15 and 20 were collected for amplification and identification of gB,gD and gI genes.The results showed that the corresponding exogenous genes were identified and the gene sequence was correct.The results showed that the exogenous genes could be stably inherited in the construction of recombinant NDV virus.Then,the heat resistance of the three recombinant viruses was evaluated.The recombinant viruses with the same HA titer were treated at 56? for 10 min,20 min,30 min,40 min,50 min and 60 min respectively.The results showed that the HA titer of the three recombinant viruses was still not less than 6log2 after 60 min treatment.LaSota strains under the same treatment conditions lost HA activity after being treated at 56? for 10 min,indicating that the three recombinant viruses all had good heat resistance.In order to evaluate the virulence of the three strains of recombinant virus,the infective ability and virulence of the three strains to SPF chicken embryos were measured on chicken embryos aged 9 to 10 days.The results showed that the EID50 of NDV/rAHR09-?/ILTV-gB,NDV/rAHR09-?/ILTV-gD and NDV/rAHR09-?/ILTV-gI were 107.83EID50/0.2 mL,108.37EID50/0.2 mL and 109.5EID50/0.2 mL,the MDT of the three recombinant viruses were all more than 120 h,which were 148 h,160 h and 159.5 h,respectively.Then,the pathogenicity of the three recombinant viruses was determined.The ICPI results were 0,0.2 and 0.1,respectively,less than 0.5,and the IVPI index was less than 0.8.These results indicated that the three recombinant viruses all met the characteristics of NDV attenuated virus and could be used as candidate ILTV vaccine strains for evaluation of immune and challenge protection.