Vaccine 27 (2009) 4630–4640 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Altered pathogenicity, immunogenicity, tissue tropism and 3′-7 kb region sequence of an avian infectious bronchitis coronavirus strain after serial passage in embryos Shengwang Liu ∗, Xiaonan Zhang, Liyang Gong, Baolong Yan, Chengren Li, Zongxi Han, Yuhao Shao, Huixin Li, Xiangang Kong Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Maduan Street, Harbin 150001, PR China a r t i c l e i n f o Article history: Received 20 January 2009 Received in revised form 22 March 2009 Accepted 25 May 2009 Available online 11 June 2009 Keywords: Avian infectious bronchitis coronavirus Pathogenicity Immunogenicity Tissue tropism Genome sequence Serial passage a b s t r a c t 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. All rights reserved. 1. Introduction Coronaviruses belong to the family Coronaviridae and the order Nidovirales, and are classified into three groups based on the absence of genetic and antigenic relationships between the species of the different groups [1,2]. They are known to cause upper and lower respiratory diseases, gastroenteritis, and central nervous sys- tem infection in a number of avian and mammalian hosts, including humans [3]. The infectious bronchitis virus (IBV) belongs to the group 3 coronaviruses. It primarily causes respiratory disease in domestic fowl, although it also replicates on epithelial surfaces of the alimentary tract, oviduct, and kidney, and is one of the most eco- nomically important pathogens in the poultry industry [4]. IBV has four essential structural proteins: the phosphorylated nucleocap- sid (N) protein, and the three membrane proteins spike (S), integral membrane (M), and small envelope (E). Although the S1 subunit of the S protein carries virus-neutralizing and serotype-specific deter- ∗ Corresponding author. Tel.: +86 451 85935065; fax: +86 451 82734181. E-mail address: