Altered pathogenicity, immunogenicity, tissue tropism and 3′-7kb region sequence of an avian infectious bronchitis coronavirus strain after serial passage in embryos

Abstract

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'-7kb 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'-7kb 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'-7kb 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.

Genomic Characteristics and Changes of Avian Infectious Bronchitis Virus Strain CK/CH/LDL/97I after Serial Passages in Chicken Embryos

“…The IBV strain Beaudette was attenuated after several hundred passages in embryonated hen's eggs [17] resulting in loss of virulence and also potential loss of immunogenicity. A similar result was observed in IBV strain CK/CH/ LHLJ/04V, the immunogenicity of which was substantially decreased after 110 passages in embryonated hen's eggs, resulting in loss of virulence to chickens [16] . However, most attenuated IB vaccine strains, such as H120, maintain their immunogenicity after adaptation to embryonated chicken eggs [12][13][14][15] , though the genomic mutations in passaged strains associated with attenuation of pathogenicity, immunogenicity, tissue tropism, and replication capacity in chicken tissues are unknown and variable, leading to differing efficacies associated with different vaccines.…”

“…The initial results (data not shown) revealed that primers N (-) contained sequences consistent with those of CK/CH/LDL/97I, and reverse transcription (RT) was therefore performed with M-MLV reverse transcriptase (Invitrogen) using the primer N (-). RT procedures were performed using 40 μl of RNA in an 80-μl reaction volume, as previously described [16] . Each cDNA fragment was PCR-amplified from the RT products, as described previously [16] .…”

“…RT procedures were performed using 40 μl of RNA in an 80-μl reaction volume, as previously described [16] . Each cDNA fragment was PCR-amplified from the RT products, as described previously [16] . PCR products were purified from agarose gels using a DNA extraction kit (Boehringer Mannheim GmbH, Mannheim, Germany) and cloned into the pMD18-T vector (TaKaRa Bio, Inc., Dalian, China), following the manufacturer's instructions.…”

“…material, see www.karger.com/doi/10.1159/000365193). RNA extraction, cDNA generation, PCR amplification, and genefragment cloning and sequencing were conducted independently for each of the two passages [16] . The 3 ′ and 5 ′ ends of the viral genomes were confirmed by rapid amplification of cDNA ends (RACE) using a 3 ′ /5 ′ RACE kit (TaKaRa Bio, Inc.), according to the manufacturer's instructions.…”

“…IB is mainly controlled by the use of live-attenuated vaccines derived from virulent viruses prevalent in a particular country or region by multiple serial passages in 9-to 11-day-old embryonated chicken eggs [12][13][14][15][16] . As a consequence of this process, the virus becomes more adapted and more highly pathogenic to the embryo, with concomitant attenuation in mature chickens.…”