Construction And Immune Efficacy Evaluation Of An Attenuated Genotype Ⅰ/Ⅶ Newcastle Disease Virus F And HN Gene Chimeric Vaccine Candidate LX-OAI4S

Newcastle disease(ND),caused by virulent strain of Newcastle disease virus(NDV),is a highly contagious avian infectious disease and hinders the development of the poultry industry.It is considered to be one of the most important avian pathogens worldwide along with highly pathogenic avian influenza virus.In recent years,surveillance of NDV shows that genotype Ⅶ NDV is still predominant at chickens and waterfowl flocks in China.In 2014,the inactivated vaccine A-Ⅶ against genotype Ⅶ NDV was issued and applied at poultry flocks in China.However,the inactivated vaccine mainly relies on injection immunization,leading to the strong animal stress and labor cost.Thus,the large-scale immunization route is essential to prevent genotype Ⅶ NDV,such as spraying and in-ovo vaccination.In this study,we selected the genotype I NDV strain LX as the backbone to construct a chimeric recombinant virus expressing F and HN proteins of genotype Ⅶ NDV,and the immune efficacy was further evaluated through different immunization methods.1.Construction and evaluation of a recombinant chimeric vaccine strain LX-OAI4S by intranasal immunizationTo obtain an ND candidate vaccine strain suitable for the epidemic strain.In this study,we successfully generated a recombinant chimeric LX candidate vaccine strain,LX-OAI4S,which harbors the ectodomain of the F protein from A-Ⅶ vaccine strain by mutating the velogenic amino acid motif at the F protein cleavage site using reverse genetics techniques,and the HN protein of velogenic JS14/12/Ch strain.The recombinant chimeric virus LX-OAI4S had intracerebral pathogenicity index(ICPI)of 0.00,mean death time(MDT)of>120 h,and 50%egg infections dose(EID50)of 10-9/0.1 mL.The recombinant virus propagated in 9-day-old specificpathogen-free(SPF)embryonated chicken eggs for nine passages showed no change in the virulence and reproductive ability.Vaccination of white leghorn chickens with LX-OAI4S and another candidate vaccine strain LX/AI4-E347K containing 106 EID50,105 EID50or 104 EID50 provided 100%protection against the velogenic NDV JS02/06.These results indicated that 104 EID50 was the minimum immune dose contributing to 100%protection in LX-OAI4S vaccination.Haemagglutination-inhibiting(HI)titers of LX-OAI4S vaccinated groups at a dose of 105 EID50 and 106 EID50 were averagely 6.30 log2 and 6.53 log2 at one week post vaccination and significantly higher than HI titers of the LX/AI4-E347K vaccinated group at the same dose,respectively.As the results implied,LX-OAI4S mediates a stronger immune efficacy than LX/AI4-E347K.The comparative protection assay between the candidate vaccine strain LX-OAI4S and commercial vaccine strains La Sota and VG/GA showed that,HI titers of the LX-OAI4S vaccinated groups were averagely 4.71 log2 at one week post vaccination and significantly higher than HI titers of the La Sota(3.43 log2)and VG/GA(2.83 log2)vaccinated groups.Moreover,virus isolation assay of the LX-OAI4S vaccinated group showed that no virus shedding(0/7)in oropharyngeal and cloacal swabs at 3,5 and 7 days post-challenge.Notably,viral shedding in the LX-OAI4S vaccinated group was significantly lower than groups vaccinated with commercial vaccine strain La Sota(2/7 in oropharyngeal and cloacal swabs,respectively)or VG/GA(3/7 in oropharyngeal swabs 1/7 in cloacal swabs,respectively)at 3 days post challenge.These results demonstrate that the LX-OAI4S strain is a valuable vaccine candidate to control ND.2.Evaluation of immune efficacy of spray vaccination with recombinant candidate vaccine strain LX-OAI4S1-day-old SPF chickens and commercial chickens were vaccinated against Newcastle disease by spray vaccination with the candidate vaccine strain LX-OAI4S and commercial vaccine strains La Sota and VG/GA at different doses.Subsequently,the vaccinated chickens were monitored up to 14 days post vaccination.The weight of all vaccinated chickens increased,but the vaccinated SPF chickens showed the weight loss compared with the control group due to immunization stress.By contrast,the weight of commercial chickens increased normally after immunization.Both SPF chickens and commercial chickens aerosol spraying with La Sota developed respiratory symptoms to varying degrees.In vaccinated groups at a dose of 2×106 EID50,aerosol spraying of 1-day-old chickens with LX-OAI4S induced the higher titer of HI than that of La Sota and VG/GA groups.Of note,one La Sota-vaccinated chicken showed neurological symptoms after challenge at two weeks post vaccination.The LX-OAI4S and VG/GA group show no clinical symptom after vaccination,and their protection rates were up to 100%.No viral shedding was detected in the oropharyngeal and cloaca swabs collected from the LX-OAI4S and VG/GA group.However,virus shedding was detected in the cloaca swabs at five and seven days post challenge with velogenic NDV in the La Sota group with the positive rate of 1/10,respectivly.In vaccinated groups at a dose of 10×106 EID50,spray immunization of SPF and commercial chickens induced the higher HI titers than that of other vaccinated groups,while one commercial chicken died in La Sota group.The clinical investigation suggested that more La Sota-vaccinated chickens showed haemorrhage from throat(SPF chicken 8/10,and commercial chickens 8/9),followed by LX-OAI4S-vaccinated chickens(SPF chicken 6/10,and commercial chickens 5/10),and VG/GA-vaccinated chickens(SPF chicken 4/10,and commercial chickens 5/10)with one chicken appearing haemorrhage from gland.The antibody titers of the LX-OAI4S increased rapidly at the first three weeks,then steadily rose reaching a peak at five weeks post immunization,and finally decreased at a slow rate.Similarly,the antibody surveillence assay indicated that the spray vaccination at 1-day old chicken induced rapid increase of HI titers druing the first three weeks and slow decrease after the fifth week post vaccination.It gave rise to high antibody titre persisting for at least 12 weeks.In summary,we constructed an ND chimeric attenuated vaccine strainLX-OAI4S.The minimum immune dose of the vaccine candidate strain LX-OAI4S was 104 EID50.Intranasal vaccination of three-week-old SPF chickens with LX-OAI4S provided 100%clinical protection,and it effectively reduced virus shedding of vaccinated chickens.Futhermore,spray vaccination of 1-day-old chicks with LX-OAI4S showed the lower influence on the weight of chicken.The vaccine candidate strain LX-OAI4S was safer and more protective than commercial vaccines.Hence,the recombinant chimeric virus LX-OAI4S is expected to be one of the vaccines used in aerosol immunization.