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-polymer ase chain reaction (RT-PCR) product of the S-l subunit of the spike peplome r gene was used to identify infectious bronchitis virus (IBV) serotypes. De generate primers CK4 and CK2, utilized previously in our laboratory, were s elected for DACS because they successfully amplify a wide range of serotype s represented by various reference strains and field isolates and the resul ting polymerase chain reaction (PCR) product contains diagnostically releva nt S-l sequences that can be used to identify the serotype of IBV. The S-l nucleotide sequences generated by DACS were aligned and analyzed with comme rcial software to determine their relationship to the S-l nucleotide sequen ces of IBV strains on deposit in the GenBank and EMBL databases. Reference strains Massachusetts (Mass) 41, Connecticut: (Conn), Arkansas (A rk) 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" compris ed of IBV reference strains, field isolates, and variant serotypes collecte d by our laboratory The DACS procedure provided high-quality and reproducib le S-1 sequence for all IBV serotypes tested, including variant serotypes t hat had not been sequenced previously. The S-1 nucleotide sequences for the amplified PCR products of reference strains Mass 41, Conn, Ark DPI, JMK, a nd DE/072/92 generated by DACS were highly homologous (>99% nucleotide iden tity) with their respective GenBank database sequences. In the unknown pane l, 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 (greater than or equal to 95.5%) after alignment with databas e sequences. In contrast, the nucleotide identities of S-1 sequences of var iant 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%, w ell below the identity values for a homologous serotype. With alignment sof tware, the identities of strains in mixtures of RNAs of two different serot ypes were not resolvable. DACS of IBV S-1 RT-PCR products will enable resea rchers to rapidly identify field strains, including new, previously unrecog nized variant virus serotypes.