| Infectious bronchitis virus(IBV)causes a highly contagious disease,which results in serious economic losses to the poultry industry around the world.Enveloped IBV belongs to the genus Coronavirus,family Coronaviridae,order Nidovirales.The most prominent of its structural proteins should be the Spike(S)glycoprotein,which consists of divergent S1 subunit and conserved S2 subunit.Previous researchers have defined particularly variable segments as hypervariable regions(HVRs)at the amino terminus of S1 subunit.There are evidences that five neutralizing peptides mainly mapped on the HVRs,and the receptor-binding domain may partly overlap with the HVRs.Thus,the HVRs possibly account for antigenicity,serotype variation and tropism.We used the strains Beaudette and ck/CH/LDL/091022 in our study.IBV Beaudette is a well-known apathogenic lab strain,which is adapted to Vero cells.IBV strain ck/CH/LDL/091022,an epidemic virulent strain isolated in China,causes the predominant gross lesions in chicken kidneys.In addition,Beaudette and ck/CH/LDL/091022 belong to different serotypes.In our study,the recombinant IBV Beaudette was recovered with the reverse genetic system.And then,we compared the amino sequences of the HVRs in Beaudette and ck/CH/LDL/091022.The three HVRs of Beaudette(HVR I,50~86 aa;HVR II,104~138 aa;HVR III,272~291aa)were separately or simultaneously replaced by the responding fragments of ck/CH/LDL/091022(HVR I,50~86 aa;HVR II,104~140 aa;HVR III,274~293aa).The responding plasmids were digested with BsmB I or Bsa I,and the fragments ligated into cDNA covering the IBV genome,which was electroporated into Vero cells.The seven recombinant IBVs were respectively named after rHVR I?rHVR II?rHVR III?rHVR I/II?rHVR I/III?rHVR I/II/III.The seven recombinant IBVs were identified,purified,titrated,and characterized with their virulence,tropism,and serotypes.Firstly,all the seven recombinant IBVs proliferated in Vero cells,but the heterogeneous HVRs may weaken their ability of adsorption during infection in vitro.The results of IFAT and RT-PCR revealed that the heterogeneous HVRs could not change the cell tropism of the recombinant IBVs.Secondly,the recombinant IBVs still shared the same serotype with Beaudette strain,but antigenic relatedness values(ARV)between the recombinant strain and Beaudette decreased with the number of the HVRs exchange increasing in general.The results of neutralization test revealed that the heterogeneous HVRs could not result in the serotypes swtiches of the recombinant IBVs.Thirdly,the infected 7-day-old SPF chickens with the seven recombinant IBVs were lack of clinical symptoms,with no mortality rate.The tracheal lesion scores of the seven recombinant IBVs were significantly lower than that of ck/CH/LDL/091022.Only low level of IBV RNA was detected in a few tracheas of the seven recombinant IBVs group.The results of in vivo infection revealed that the heterogeneous HVRs did not change the tissue tropism and pathogenicity of the seven recombinant IBVs.Finally,the vaccinated chickens challeged with ck/CH/LDL/091022 could quickly produce IBV specific antibodies.The viral load of tracheas and kidneys in vaccinated birds with the seven recombinant IBVs were significantly lower than that of PBS vaccinated birds.The results of vaccination-challenge tests revealed that the recombinant IBVs could provide protection against the disease.This study uncovered the functions of HVRs,and explored to develop a vaccine candidate,and all of the information will contribute to our further understanding of IBV prevention and control. |
PDF Full Text Request