Construction Of An Infectious Clone System For A Genotype Ⅶ Newcastle Disease Virus Strain And Its Preliminary Application In Basic And Applied Research

Newcastle disease(ND)is a highly infectious disease of poultry caused by Newcastle disease virus(NDV),which can cause damage to the respiratory system,nervous system,digestive tract and internal organs,with a lethal rate of 100%.Since the outbreak of Java Claw in India and New Town in Britain in 1926,NDV has caused four pandemics in the world,resulting huge economic losses to the poultry industry.Since the third pandemic,the ND vaccine began to be widely used worldwide.The first was the La Sota vaccine of type II.Later,vaccines developed based on Mukteswar strains,Clone 30 strains,Hitchner B1 strains,F strains,and ZJI strains gradually became available.The widespread use of vaccines has effectively suppressed the large-scale outbreak of ND globally.Entering the 1990 s,genotype VII NDV has gradually become the main epidemic strain worldwide.Because the genotype of the immunization vaccine does not match the genotype of the epidemic strain,it sometimes causes endemic ND outbreaks.Subsequently,the reverse genetic system of negative-strand RNA viruses was successfully established.The genotype VII ND vaccine was quickly developed and widely used with the help of the reverse genetic platform.It has been more than 20 years since Peeters first constructed infectious clones of NDV by reverse genetics in1999.The technique has become increasingly mature.The establishment of a reverse genetic platform has not only accelerated the development of vaccines against epidemic strains,it also makes research on NDV more convenient and in-depth.One NDV strain namely DHN3 was isolated in our laboratory in 2011 and identified as a gene VII type.In this study,an infectious clone for DHN3 was constructed using p BR322 plasmid as a vector and a p XJ40-DE3 plasmid was constructed instead of using fowlpox virus to express the T7 RNA polymerase,and the recombinant virus r DHN3 was rescued in BHK-21 cells.Subsequently,we substituted the base sequence from amino acids 112 to 117 in the coding region of the DHN3 F gene with the corresponding sequence from the La Sota strain which contains a cellular protease fusion cleavage site to obtain the attenuated strain r DHN3-m F.Furthermore,in order to study the role of the NP gene in NDV infection and replication,we constructed r DHN3-m NP which contains a modified NP gene with minimal codon usage,by using a single codon for majority of amino acid in the NP gene.r DHN3-m F and r DHN3-m NP have similar growth characteristics as the original strain DHN3.Infection of one-week old SPF chickens with DHN3,r DHN3-m F,r DHN3-m NP,and La Sota allantoic fluid live virus at 106 EID50 showed that all chickens died in the DHN3 and r DHN3-m NP groups 4days after infection.Swelling and bleeding in the heart,liver,etc.The flocks in the r DHN3-m F,La Sota and control groups were energetic,feeding and excretion were normal.Visceral examination of chicks in the r DHN3-m F,La Sota,and the control groups one week after infection demonstrated that the r DHN3-m F and control groups were normal;individual chicks in the La Sota group showed thymus and liver swelled and congested.Blood was collected at 7 d,14 d,and 21 d,respectively,after infection,HI testing showed that birds in the r DHN3-m F and La Sota groups produced NDV antibodies.The r DHN3-m F inactivated vaccine was then used to immunize one-week old SPF chickens.After three weeks of immunization,the flocks were challenged with the virulent DHN3,showing 100% protection.Using DHN3 and La Sota as antigens to detect antibody titers of r DHN3-m F inactivated vaccine groups revealed that immunized chickens produced high levels of effective antibodies.Nine to eleven day-old SPF chicken embryos were infected with DHN3,r DHN3-m F and r DHN3-m NP at 1000 PFU,respectively and the effects on chicken embryo immunerelated factors were detected by q PCR.The results showed that the virus infection of embryos caused an increase in the expression of type I interferon,and the type I interferon feedback regulators NLRC5 and SOCS1 were up-regulated simultaneously.Mutant strains could induce IFN-β upregulation faster in chicken embryos compared to that of wildtype DHN3.The DHN3 strain could induce higher expression of antiviral factors such as MOV10 and RSAD2(viperin)than others.For the HLA gene that was most up-regulated in this experiment,the r DHN3-m NP showed the highest up-regulated among the three.We believe that these differences in induction of host antiviral gene expression among wild type,r DHN3-m F and r DHN3-m NP may related to the different pathogenic potential of these viruses.Further exploration of these observations would be required.