Complete Genome Sequence of Infectious Bronchitis Virus Strain JP/KH/64, Isolated in Japan

ABSTRACT Here, we report the complete genome sequence of infectious bronchitis virus (IBV) strain JP/KH/64, which is the reference strain for the JP-I genotype in Japan. This information should be useful for an in-depth understanding of the evolution of the JP-I genotype.


GENOME SEQUENCES
nucleocapsid (N) protein. Among these proteins, the S1 glycoprotein is associated with virus attachment and is a major target of neutralizing antibodies in chickens (2)(3)(4).
In Japan, seven genotypes of IBV (JP-I, JP-II, JP-III, JP-IV, Mass, Gray, and 4/91) have been confirmed, based on the partial nucleotide sequence of the S1 gene (5,6). Analysis of genes other than S1 revealed that various recombinant viruses are also prevalent in Japan (6). A complete genome analysis of the IBV is important for understanding the epidemiology of recombinant viruses. To understand the epidemiology of variant IBV in Japan, the complete nucleotide sequence of the major genotype JP-I strain was determined.
FIG 1 Phylogenetic tree based on the complete S1 glycoprotein gene of IBV. Nucleotides 20368 to 21978 (1,632 bases) of the S1 gene of IBV Beaudette (GI-1) (GenBank accession number NC_001451) were subjected to phylogenetic analysis. The phylogenetic tree was generated using the neighborjoining method in MEGA 7 (14) with 1,000 bootstrap replications. All tools were run with default parameters unless otherwise specified. Horizontal distances are proportional to the minimum number of nucleotide differences required to join nodes and sequences. The genotypes of IBV were defined by Valastro et al. (10). The JP/KH/64 strain is indicated with a black circle.
The JP/KH/64 strain was isolated from chickens with respiratory symptoms in 1964 in Japan and was first confirmed as the JP-I genotype in Japan (5). The virus was grown in primary chicken kidney cell cultures prepared from 4-to 10-week-old chicks, and the infected culture fluids were harvested. Viral RNA was extracted from infected culture fluids using a QIAamp viral RNA minikit (Qiagen, Hilden, Germany), and random hexamer primers were used for cDNA synthesis (6). Specific primers for genome sequencing using the Sanger method were designed based on previous studies (7,8) and on the sequences obtained from amplicons (Table 1). Both the 39 and 59 termini of the genome were determined using the rapid amplification of cDNA ends (RACE) technique.
IBV is classified into six main genotypes (GI to GVI), comprising 32 viral lineages (lineages 1 to 32), based on the complete nucleotide sequences of the S1 glycoprotein and its typical geographical distribution (10). Phylogenetic analysis based on the complete coding sequence of the S1 gene reconfirmed that JP/KH/64 is a member of the GI-18 lineage, clustering in one group with the lineage prototype strain JP8127 (GenBank accession number AY296744) (87.6% nucleotide identity), which was isolated in Japan (11) (Fig. 1). Because the JP-I genotype has been reported in China, Taiwan, and Japan (12,13), this information about the JP/KH/64 strain might be important for the understanding of IBV evolution in Eastern Asian countries.
Data availability. The genome sequence of IBV strain JP/KH/64 was deposited in GenBank under accession number LC634083.