In this study, we attenuated a Chinese LX4-type nephropathogenic infectious bronchitis virus (IBV) strain, CK/CH/LHLJ/04V, by serial passage in embryonated chicken eggs. Based on sequence analysis of the 3′-7 kb region, the CK/CH/LHLJ/04V virus population contained subpopulations with a mixture of genetic mutants. The titers of the virus increased gradually during serial passage, but the replication capacity decreased in chickens. The virus was partially attenuated at passage 40 (P40) and P70, and was fully attenuated at P110. It lost immunogenicity and kidney tropism at P110 and P70, respectively. Amino acid substitutions were found in the 3′-7 kb region, primarily in the spike (S) protein. Substitutions in the S1 subunit occurred between P3 and P40 and all subpopulations in a virus passage showed the same substitutions. Other substitutions that occurred between P70 and P110, however, were found only in some subpopulations of the virus passages. A 109-bp deletion in the 3′-UTR was observed in most subpopulations of P70 and P110, and might be related to virus replication, transcription and pathogenicity. The changes described in the 3′-7 kb region of the virus are possibly responsible for virus attenuation, immunogenicity decrease and tissue tropism changes; however, we cannot exclude the possibility that other parts of the genome may also be involved in those changes. © 2009 Elsevier Ltd.
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