key: cord-0004924-frluobkw
authors: Wang, C. -H.; Tsai, C. -T.
title: Genetic grouping for the isolates of avian infectious bronchitis virus in Taiwan
date: 1996
journal: Arch Virol
DOI: 10.1007/bf01718291
sha: bf9c53277b382c09c854fed08730eff384073bdd
doc_id: 4924
cord_uid: frluobkw

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.

Avian infectious bronchitis virus (IBV) causes a highly contagious respiratory disease in chickens which is economically devastating to farmers who raise birds under intensive conditions [10] . The isolation and identification of IBV isolates throughout Taiwan are important, because vaccines are selected based on the serotypes of IBV in specific geographic areas. Traditionally, IBV serotypes are identified by the virus-neutralization (VN) test which is labor-intensive and time-consuming. However, ambiguous results are sometimes obtained using a serological approach [8, 17] . Recently, restriction fragment length polymorphism (RFLP) and sequencing have offered more sensitive and specific methods for identifying IBV strains [2] [3] [4] [5] [6] 16] . All known IBV viruses contain three major virus-encoded structural proteins which include the surface projection glycoproteins, the membrane protein, and the nucleocapsid protein [9, 10] . The VN

Rapid RNA extraction was conducted on allantoic fluid from uninoculated eggs and from eggs inoculated with Newcastle disease virus (A970, National Taiwan University), Egg drop syndrome virus (PRIAH), fowl adenovirus type I (PRIAH), infectious bursal disease virus (PRIAH) and infectious laryngotracheitis virus (Vaccine strain, Intervet, Holland). Extraction of RNA and RT-PCR were performed using the same methods described above.

Five restriction endonucleases, BsoFI, DdeI, MboII, AluI, and RsaI, were chosen based on the 22 published IBV sequences using the Mepdraw program (Lasergene). The predicted PCR products were equally divided into five tubes, and digested with each enzyme (BioLabs, Beverly, MA, USA) under the conditions recommended by the enzyme supplier. The restriction fragment patterns were observed following electrophoresis on a 2.5% metaphor (FMC Bioproducts, USA) + 1% pure agarose gel (BBL) at 100 V constant voltage. The 25 Taiwan isolates were grouped according to their RFLP patterns.

The amplified DNA was amplified again using the same primers (C2U-C3L) to obtain pure PCR products (secondary PCR). The secondary PCR products were sequenced by the dideoxynucleotide method using Taq DyeDeoxy Terminator Cycle Sequencing kit (ABI 3370, Applied Biosystems, USA). Four kinds of fluorescent dye-labeled ddNTPs were added with a single-strand primer. The sequences was analyzed by an automatic DNA sequencer (Applied Biosystems). Both strands C2U-C3L PCR products were sequenced for each isolates to ensure the accuracy of results. The Megalign program (Lasergene) was used to align the nucleotide sequences and to generate a similarity plot. If the identity was less than 80%, isolates were classified into different groups (I and II). A phylogenetic tree was drawn using the Megalign program (Lasergene).

By using the Clustal m e t h o d in the Megalign p r o g r a m (Lasergene), the 22 published IBV strains in G e n B a n k and E M B L were divided into 4 distinct genetic groups, i.e. M a s s , American, E u r o p e a n and D u t c h when analyzing the - ND Not done. + PCR product positive. -no PCR product. S1 primers, PCR product including the whole S1 gene [12] . $2 primers, PCR product locating the N-terminus of $2 gene [13] . M-N primers, PCR product including the junction part of M and N genes [1] . N primers, PCR product including N gene [18] . C2U-C3L primers, a new set of primers at the N-terminus of S1 gene w h o l e S1 gene as well as the C 2 U -C 3 L (nt 

The RFLP patterns of the 4 reference strains generated by digestion with BsoFI, DdeI, MboII, AluI, and RsaI are shown in Fig. 2 . The fragments predicted from the published sequences in GenBank is shown in Table 2 . There was only one apparent exception noted in lane 5 of Fig. 2A where MboII was supposed to generate 3 fragments, 54, 64, and 110 bp but only 2 bands were seen (110, 60 bp). Apparently the two smaller fragments migrated together. Figure 2 and Table  2 summarize the RFLP patterns of the 4 reference strains by showing the presence or absence of cleavage sites in each strain.

The RFLP pattern of one Taiwan patterns of the other 24 T a i w a n isolates in this study differed from those of the C 2 U -C 3 L S1 gene of the 4 published strains described above plus 18 other published strains whose sequences were analyzed by the Megalign p r o g r a m (Lasergene). The R F L P of these strains were c o m p a r e d and divided into 8 R F L P patterns (Table 3 and The PCR product is cut by MbolI into 2 identical fragments. Only one band is found in 5 except the original uncut PCR product (these data are given in Table 4 ) The R F L P pattern of isolate A 1928 was the same as that of A 1967 and therefore the two isolates were considered to be the same R F L P pattern (WTI-1 Table 3 ). The R F L P patterns of isolates A1121, A1210, A1952, A1980, A2012 and A2137 were the same, and were quite different from the isolates in WT1 and grouped into WT2 (Fig. 3D) .

Following the grouping of IBV isolates by PCR-RFLP, t2 representative isolates were selected from 8 R F L P patterns for direct sequencing (with "a" in Table 3 ). The length of the PCR products of A1211, A1960, A1953, A1955, A2003, A2054, A1967 was 231 bp and that of All71, A1916, A1980, A2012, and A 1121 was 228 bp. The sequences of the 12 isolates are shown in Fig. 4 . The sizes of the fragments generated by restriction endonucleases are shown in Table 4 . Since MboII cut the PCR product into 2 identical fragments (114 bp), only one band was found in the PCR product from WT2. (Fig. 3D ).

The similarity in Taiwan isolates ranged from 65.4% to 99.6% ( Table 5 ). The sequence relationships of the HVR I were shown in a phylogenetic tree (Fig. 5) . The sequence of isolate A1916 was the same as the Mass serotype. Another 11 

Due to high sequence variability between different IBV strains, it has been difficult to design PCR primers that can be reliably used to monitor all IBV isolates, especially in the S1 gene that contains hypervariable regions. In this study, we report a set of olignucleotide primers that is capable of hybridizing to the genome of all known IBV strains. The primers have now been used successfully on 4 known strains and 25 Taiwan isolates. These primers were intended to amplify a portion of the IBV genome that encodes the viral coat protein providing the antigenic determinations. This region of the S1 gene is hypervariable and apparently mutates often enough to thwart efforts to create a universal effective vaccine. The 24 isolates are related distinctly and are different from all known strains of IBV (Mass, American, European and Dutch).

The immunogenicity of Taiwan isolates needs further study by comparing with reference IBV strains. In the past, the 22 published IBV strains have been divided into 4 groups, i.e. Mass, American, European and Dutch [15] . The results from this study are entirely consistent with that classification. The strains within the Mass, European and Dutch groups are at least 95% homologous. The C2U-C3L region (PCR oligonucleotide primers in Materials and methods) was chosen for the study reported here because phylogenetic results from the sequences in this region match those from the whole S1 gene. The 24 Taiwan isolates differ from those published foreign strains and are, therefore variant strains. Since the 24 isolates have been divided into 8 RFLP patterns it is unlikely that the IBV isolates in Taiwan have evolved from the same origin. However, the nature of their evolution is totally unknown and remains to be studied. For this, it will be necessary to type foreign IBV isolates by PCR-RFLP using the same primers (C2U-C3L) so that they can be compared to the Taiwan isolates.

The variation of IBV may also be attributed to recombination following co-infection of two distinct strains [14] . Several investigators have characterized the isolates obtained from IBV outbreaks, and showed that they had a strong relationship with the vaccine strain, suggesting that the prevalent isolates may have originated by recombination with live vaccine strains [14] . By contrast, our present observation that the 24 isolates are different from the reference strains, including current vaccine strains, suggests that they are not derived from live attenuated vaccines.

IBV causes severe poultry losses in young broiler populations despite the use of Mass serotype vaccines in Taiwan. An accurate determination of which serotypes or subtype are causing field outbreaks would be valuable to the poultry industry. Although attempts have been made to classify IBV isolates by serologic methods, it has become apparent that serology alone in not adequate [8, 17] . PCR-RFLP is a convenient method to classify new isolates, since the C2U-C3L primer set detects all Taiwan isolates. It is necessary to survey whether the prevalent IBV isolates are similar to or different from current vaccine strains. The pathogenicity and immunogenecity of different RFLP patterns of the Taiwan isolates needs further study. Since these isolates are quite different from vaccine strains used currently, it is necessary to develop a local vaccine in order to control IBV infection in Taiwan.

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We are grateful to Dr. William L Miller, WNR Professor of Biochemistry at North Carolina State University, USA for reviewing our manuscript. This work is financed by the National Science Council, ROC (NSC85-232 t-B002-112).

 5 6.5 ' 5. 6 Received October 2, 1995