Identification of Avian Infectious Bronchitis Virus by Direct Automated Cycle Sequencing of the S-1 Gene

Direct automated cycle sequencing (DACS) of a reverse transcription-polymerase chain reaction (RT-PCR) product of the S-1 subunit of the spike peplomer gene was used to identify infectious bronchitis virus (IBV) serotypes. Degenerate primers CK4 and CK2, utilized previously in our laboratory, were selected for DACS because they successfully amplify a wide range of serotypes represented by various reference strains and field isolates and the resulting polymerase chain reaction (PCR) product contains diagnostically relevant S-1 sequences that can be used to identify the serotype of IBV. The S-1 nucleotide sequences generated by DACS were aligned and analyzed with commercial software to determine their relationship to the S-1 nucleotide sequences of IBV strains on deposit in the GenBank and EMBL databases. Reference strains Massachusetts (Mass) 41, Connecticut (Conn), Arkansas (Ark) DPI, JMK, and DE/072/92 were initially tested by DACS to establish the feasibility of the procedure. The DACS procedure was further evaluated with a panel of "unknowns" comprised of IBV reference strains, field isolates, and variant serotypes collected by our laboratory. The DACS procedure provided high-quality and reproducible S-1 sequence for all IBV serotypes tested, including variant serotypes that had not been sequenced previously. The S-1 nucleotide sequences for the amplified PCR products of reference strains Mass 41, Conn, Ark DPI, JMK, and DE/072/92 generated by DACS were highly homologous (>99% nucleotide identity) with their respective GenBank database sequences. In the unknown panel, the nucleotide identities of the DACS S-1 sequences of field isolates of serotypes previously identified by virus neutralization were also found to be very high (> or = 95.5%) after alignment with database sequences. In contrast, the nucleotide identities of S-1 sequences of variant serotypes 37, 3330, and PA/1220/98 and reference strain Clark 333, for which database sequences were not available, ranged from 27.7% to 73.8%, well below the identity values for a homologous serotype. With alignment software, the identities of strains in mixtures of RNAs of two different serotypes were not resolvable. DACS of IBV S-1 RT-PCR products will enable researchers to rapidly identify field strains, including new, previously unrecognized variant virus serotypes.