Study On Variation Of S1 Gene Of IBV During Serial Passages In CEK Cell Cultures

In this experiment, we study the chronological order genotypic changes in the spike protein ofstrain M41 of avian infectious virus (IBV) during its adaptation to a primary chick embryo kidneycell (CEK).We propagated the M41 strain of IBV embryonic passage (E6) for thirty passagesserially on CEK cell. The S1 gene of virus from E6, CEK cell passage1(F1),4(F4),10(F10),15(F15),20(F20),25(F25),30(F30) was cloned into pMDI8-T vecter andtransformed into E coli DH5αstrain, the positive recombinants selected randomly were sequenced,the homology of nucleotide and deduced amino acid sequence were analysed by DNAstar andDNAsis software, the results were as follows:The homology of nucleotide and deduced amino acid sequence of S1 gene between E6 anddifferent passages in CEK cell were very high, It was as high as 99.4％and 98.7％,respectively. Inaddition, The homology of S1 gene among different passages in CEK cell was very high, too.Analysis of phylogenetic tree of S1 gene indicated that E6, F1 and F4 had closer relationship andwere classified as the same phylogenetic group; There were 27 nucleotide mutation in differentpassages in CEK cell, causing 16 amino acid substitutions in S1 protein. Among these mutation,variation distributed over the whole S1 gene and any passage on CEK cell. Almost mutation israndom and unlimited to some domain in S1 gene. Nucleotides at position 1020,1465 have gradualchange. At these positions, T residue substituted G residue and T residue substituted A residue,respectively. Correspondingly, amino acid at position 340,489 of S1 protein Phe taken the place ofLeu and Try replaced Asn from F10 to F30. It is obvius that the dominant CEK cell-adapted Virusmay be appeared between F4 and F10.These suggested that the dominant CEK cell-adapted Viruswas favoured to T,G at S1 gene position 1020 and 1465 and to Phe, Try at S1 amino acid position340 and 489, respectively.