In order to differentiate recent isolates of avian infectious bronchitis virus (IBV) in Taiwan, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and direct sequencing methods were used to type 25 IBV Taiwan isolates. Two conserved sequences that flank the hypervariable region I (HVR I) in the N-terminus of S1 protein gene were chosen as primers. Sequences of 228-231 base pairs (bp) were amplified by PCR from 25 Taiwan isolates and 4 reference strains (H120, Conn, JMK, Holte). PCR products were digested with 5 restriction endonucleases, BsoFI, DdeI, MboII, AluI, RsaI, and different IBV isolates were grouped according to their RFLP patterns. The RFLP patterns of the 4 reference strains in this study matched the published sequences in GenBank. Except 1 vaccine strain, the other 24 Taiwan isolates were different from these 4 and 18 other IBV strains whose sequences were published. The data from PCR-RFLP and sequencing of IBV genomes showed that the 24 Taiwan isolates can be divided into 2 distinct groups, I and II. Seven RFLP patterns are identified in group I and only 1 in group II. 1678 C.-H. Wang and C.-T. Tsai antibodies that normally form the basis for comparison of IBV isolates are induced largely by the N-terminus of the S1 protein [7, 9, 11-]. In the present study, we used reverse transcription polymerase chain reaction (RT-PCR) coupled with restriction fragment length p o l y m o r p h i s m (RFLP) of the R T -P C R products to classify our isolates. Using these techniques, we present a convenient m e t h o d for identifying the R F L P patterns of IBV isolates in Taiwan which are different from those of k n o w n IBV strains. Materials and methods Twenty-five Taiwan isolates of IBV were used in this study. and V1397. Four sets of primers successfully used by other [1, 12, 13, 18] and one new set of primers were used in this study. The published primers included S1 [12], $2 [13], M-N [-1], and N primers [18] . For the new primers, the S1 sequence of the 22 published IBV were analyzed using the Clustal method to find conserved sequences. The new primers (C2U-C3L) were located in the N-terminus of the S1 gene which contains hypervariabte region I (HRV I, nucleotides, nt 142 320); this region is reponsible for induction of antibodies to the IBV that have been the basis for defining of different IBV serotypes [9, 11] . The sequences and locations of the new primers used for the PCR were C2U: TGGTT GGCAT TTACA CGGGG (114-133) and C3L: CAATG GGTAA CAAAC AC (341-325, as in Beaudette strain). Viral RNA was extracted as described [t2] with some modification. Briefly, guanidine thiocyanate (Sigma), and NaOAC were added to allantoic fluid, and extracted with phenolchloroform-isoamylalcohol. The RNA was further precipitated in isopropanol (Merck) and resuspended in alcohol and stored at -20 °C until it was used in RT-PCR. Genetic typing of infectious bronchitis virus in Taiwan 1679 All glassware and double distilled water (DDW) were treated with 0.1% diethyl-pyrocarbonate (Sigma) for removing RNase contamination. The RT-PCR was accomplished in one step. The mixture included 10 ~tl of pfu polymerase 10 x buffer (Stratagene, USA), 8 gl of 2.5 mM dNTP (Stragene), 0.4 gl of 40 U/gl RNasin (Promega), 0.3 gl RTase (AMV, Promega), 1 ~tl primers (20 ~tM), 0.8 gl of 2.5 U/gl pfu polymerase (Stragene), 60 gl DDW and 20 gl viral RNA. The RT-PCR was conducted in a FTS-960 thermal cycler (Corbett Research, Australia) where the RT was performed at 42 °C for 1 h. PCR was then performed during 35 cycles of denaturation at 94 °C for 30 sec, annealing at 45 °C for 1 rain, and polymerization at 72 °C for 1 rain. The final polymerization step was conducted at 72 °C for 7 rain. The PCR products were analyzed on a 2% gel containing ethidium bromide (0.5 gg/ml), and the amplified DNA was detected using an ultaviolet transilluminator.
Output: Genetic grouping for the isolates of avian infectious bronchitis virus in Taiwan