Construction And Immune Efficacy Evaluation Of Recombinant Newcastle Disease Virus LaSota Expressing The LX4 Genotype Infectious Bronchitis Virus S1 Protein

Avian infectious bronchitis(IB)caused by avian infectious bronchitis virus(IBV)is an acute and highly contagious respiratory disease of chickens,causing enormous economic loss in the poultry industry.The first IBV serotype known as Massachusettss(Mass)type was identified in 1930 s,numerous serotypes and variants have been isolated worldwide since then.The poor cross-protection among IBV strains belonged to different serotypes or genotypes has been revealed,which complicated the control of IB.Recently,the prevalence of LX4 genotype IBV has been reported in many parts of the world and represents a persistent threat to poultry industry in many countries.Although the Mass type vaccines have been extensively used for IB control in China,LX4 genotype IBVs still could be consistently isolated from vaccinated chicken flocks,suggesting that the currently used Mass type vaccines could not be able to provide effective protection against these epidemic isolates.And it is urgent to develop novel IBV vaccines in order to better control this disease.In order to develop a novel IBV vaccine against LX4 type,a recombinant Newcastle disease virus(r NDV)expressing S1 gene of LX4 type IBV ck/CH/LDL/091022 based on La Sota vaccine strain named r La Sota-S1 was generated via reverse genetics.The growth dynamics of r La Sota-S1 in eggs was similar to that of the parental La Sota strain,indicated that the insertion of IBV S1 did not affect the replication of vector virus.The MDT and ICPI values of the r La Sota-S1 were165.3h,0.00,while the MDT and ICPI values of the r La Sota-S1 were 100 h,0.19,which indicated that the insertion of IBV S1 slightly decreased the virulence of the parental virus.The results of indirect immunofluorescence assay(IFA)and Western blot demonstrated that and S1 protein could be expressed in r La Sota-S1 infected cell and incorporated into the virions.To evaluate the protection efficacy of r La Sota-S1,r La Sota-S1 and La Sota were used to vaccinate SPF chickens;serums were isolated once a week.HI assay demonstrated that both r La Sota-S1 and La Sota induced NDV HI antibody.ELISA assay indicated that vaccination with the r La Sota-S1 strain induced IBV-specific antibody.At 21 days post-vaccination(dpv),chickens were challenged with the genotype Ⅶ virulent NDV ck/CH/LHLJ/1/06 strain and the LX4 type IBV ck/CH/LDL/091022 strain.The results showed that after challenge with virulent NDV,all chickens immunized with the La Sota and r La Sota-S1 strains were protected completely,and no clinical signs and mortality were observed.In contrast,all chickens in the control group died at 4 days-post challenge(dpc).Only 10% of the oral swabs from birds of both vaccinated groups were positive for NDV detection at 4 dpc,and no virus shedding was detected after 8 dpc.After challenge with IBV,oral swabs were collected and the IBV RNA was detected via real time PCR.The rate of viral shedding in r La Sota-S1 vaccinated group at 5,10,15,20 dpc were 80%,50 %,33.3 %,and 16.7 %,respectively.In the control group the rate of viral shedding at 5,10,15,20 dpc were 100%,66.7%,66.7%,and 33.3%,respectively.The rate of viral shedding in r La Sota-S1 vaccinated group was significantly lower than the control group.A 40% mortality rate was observed in the vaccinated chickens,while a 70% mortality rate was observed in the control.Since only partial protection was obtained after vaccinated with r La Sota-S1 single time,the immune program was further adjusted.A booster vaccination was performed 14 days after the first vaccination using the same procedure.Peripheral blood lymphocytes(PBLs)and sera were collected once a week.The T lymphocyte subsets in the PBLs were analyzed by flow cytometry,and IBV antibodies were detected by ELISA.The percentages of CD3+CD4+ and CD3+CD8+ T lymphocytes in the vaccinated group were significantly higher than that of control group.Moreover,after the boost vaccination,the percentages of CD3+CD4+ and CD3+CD8+ T lymphocytes were significantly increased.These results indicated that r La Sota-S1 can induce cellular immunity.The ELISA assay showed that the level of IBV antibody was also significantly increased.Chickens were challenged with virulent IBV at one week after the boost vaccination,and oral swabs were collected.The viral loads in swabs were determined via real time PCR.The results showed that in comparison with single vaccination,the viral shedding rate in oral swabs from chickens boost vaccinated with r La Sota-S1 at 5,10,15,20 dpc were 66.7 %,35.7 %,14.3 %,7.1 %,respectively,significantly lower than those of the control group.In addition,five chickens from each group were killed humanely at 5 dpc,and tissue samples were collected.The viral loads in tissues were evaluated via real time PCR.The results indicated viral loads in all tissues from the prime-boost vaccinated group were significantly lower than those in the control groups.These results indicated that the protection efficacy of boost vaccination against virulent IBV challenge was significantly higher than single vaccination.The clinical protection rate was 93.3%.