Retriction Fragmentlength Polymorphism And Sequence Analysis Of Avian Infectious Bronchitis Field Isolates

Three sequences of 228bp,602bp, 1720bp that contains the partial or whole of S1 glycoprotein gene of IBV were amplified by polymerase chain reaction(PCR).five of six reference strains(Mu, Holte, Arka, Conn, Gray, T) and six of eight field isolates (138, Yi, Guang32, Yun, 14Hu, Am, J7, ZhangIBV) were amplified successfully using the primers of 602bp fragment;two reference strains ( M41, Conn) and two field isolates(14Hu, Am) were amplified successfully using the primers of 228bp fragment;only the reference strain(M41) and the field isolate strain 138 were amplified using the primers of 1720bp fragment. So we choose the 602bp fragment of PCR products and digest it with three restricton enzyme(RE)Alu I, Taq I, Afa I respectively ,six reference strains were grouped into three patterns according to the restriction fragment length polymorphism (RFLP) analysis :(1)the pattern of Mn,digested positively with Alu I, Taq I and Afa I ;(2)the pattern of Ark, Gray, Conn, digested positively with both Alu I and Afa I but negatively with TaqI ,(3)the pattern of T, digested positively with both Taq I and Afa I ,but negatively with Alu I .similarly, six field strains were grouped into two patterns according to the same restriction fragment length polymorphism analysis :(1)the pattern of M41 (Guang32, Am, J7, ZhangIBV),;(2)the pattern of Ark, Gray, Conn(138, Yi);It suggest that there are multiple serotypes of IBV in our country,but the respiropathogeic IBV represented by M41 is very popular,accounting for 50%, the nephropathogenic IBVrepresented by Yi strain also have certain popularity,making up 25%. The 602bp fragment of Yi isolate is sequenced and analysised by DNAstar ,compared with the relevant sequences of Ark, Gray, Conn which is in the same RFLP pattern and M, which is in the different RFLP pattern, the Yi strain may be a varriant of nephropathogenic IBV caused mainly by piont mutation and partially by gene recombination, According to the analysis of amino acids homogeneity ,M41, H52, H120 were mostly homogenic,with the homogeneity of 95.8%, 97%, 98.8% respectively;Yi isolate has the highest homogeneity with Ark, Gray (the homogeneity is 91.6%, 88% respectively) and a lower homogeneity of 85.6%, 86.2%, 86.2% respectively with M41,H52, H120-therefore, it can be concluded that Yi isolate may come from the serotype of Ark-1529-29.This study can provide practical value for diagnosis and differentiation of IBV,it also can provide reference data for prevention against IBV and the selection of vaccine strains.