Molecular characterisation of very virulent infectious bursal disease viruses in Taiwan

Research in Veterinary Science 2001, 70, 139–147 doi:10.1053/rvsc.2001.0450, available online at http://www.idealibrary.com on

Molecular characterisation of very virulent infectious bursal disease viruses in Taiwan H. J. LIU*, P. H. HUANG, Y. H. WU, M. Y. LIN, M. H. LIAO Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan, ROC SUMMARY The very virulent infectious bursal disease virus (vvIBDV) RNA in the bursa of Fabricius and spleen from experimentally infected chickens or field samples was detected by in situ hybridisation (ISH) with subsequent reverse transcription (RT)-polymerase chain reaction (PCR) and sequence analysis. The VP2 gene of vvIBDV was detected by ISH in infected chicken tissues with a cloned digoxigenin (DIG)-labelled cDNA probe. To verify ISH, RT-PCR was used to amplify two 643- and 500-base pair fragments on the VP2 gene of IBDV in the bursa of Fabricius. With all isolates, two cDNA fragments of 643 and 500 bp long, respectively, were generated as expected and further confirmed the specificity of ISH. Analysis of the hypervariable region (HVR) of the VP2 gene revealed that a serine-rich heptapeptide SWSASGS located at amino acids 326–332 was conserved in recent Taiwanese strains, and two amino acid substitutions were found in the classical Taiwanese strains at positions 330M and 331W. Three amino acids were unique to the vv strains at positions 222A, 256I and 294I, compared with classical and variant strains. Sequence and phylogenetic analysis showed that the recent Taiwanese strains were closely related, very similar to vvIBDVs from Europe, China, Japan, and Africa, and distantly related to the Taiwanese classical strains. © 2001 Harcourt Publishers Ltd

INFECTIOUS bursal disease (IBD), caused by a birnavirus, has spread to essentially all major poultry producing areas of the world. Infectious bursal disease virus (IBDV) causes a variety of disease syndromes in young chickens (3–6 weeks old), ranging from loss of feed efficiency to ablation of the humoral immune response (Winterfield et al 1972). At least two serotypes of the virus are currently recognised (McFerran et al 1980, Jackwood et al 1982); the known serotype 1 viruses are pathogenic only in chickens. Pathogenic serotype 2 viruses have not been isolated (Cummings et al 1986). IBDV has a dsRNA genome that divided into two segments (A and B) (Muller et al 1979). The A-segment (3.2 Kb) contains two partly overlapping open reading frames (ORFs). The first, smallest ORF encodes the non-structural viral protein 5 (VP5, 17 KDa; Mundt et al 1995). The second ORF encodes a polyprotein (110 KDa), which is autocatalytically cleaved into pVP2 (48 KDa), VP4 (28 KDa), and VP3 (32 KDa) (Hudson et al 1986, Muller and Nitschke 1987). pVP2 is further processed into VP2 (38 KDa), probably resulting from site-specific cleavage of pVP2 by a host cell encoded protease (Kibenge et al 1997). The VP2 contains the antigenic regions responsible for neutralising antibodies and serotype specificity (Azad et al 1987, Becht et al 1988, Fahey et al 1989). The epitopes recognised by neutralising antibodies are conformational and have been mapped to the hypervariable region (HRV) of VP2 between deduced amino acids 206–305 (Baylisis et al 1990, Heine et al 1991). The segment B (2.8 Kb) contains one large ORF, encoding the

VP1 (91 KDa), the putative dsRNA polymerase (Spies et al 1987, Morgan et al 1988). Demonstration of the virus in bursal tissues can be accomplished by isolation in tissue culture, embryonated chicken eggs, electron microscopy, immunological methods, and molecular-based techniques. Polymerase chain reaction (PCR) (Saiki et al 1985) and molecular hybridisation techniques (Nuovo et al 1994, Liu and Giambrone 1997, Liu 2000, Liu et al 2000) are finding applications in many areas of diagnostic testing and provide increased sensitivity with less labour and time (Tenover 1988). The acute form of IBD caused by very virulent IBD virus (vvIBDV) was first described in Europe at the end of the 1980s (Chettle et al 1989, van den Berg et al 1991). This new form of the disease was then described in Japan in the early 1990s (Nunoya et al 1992, Lin et al 1993), and it has rapidly spread all over Asia and to other major parts of the world (Cao et al 1998). Little is known about genetic and antigenic characteristics of vvIBDV in Taiwan. In an attempt to understand the current IBDV situation and to investigate how new Taiwanese isolates evolve, we developed in situ hybridisation (ISH) and reverse-transcription (RT)-PCR to detect vvIBDV in chicken tissues and compare their nucleotide and deduced amino acid sequences with previously published sequences to study antigenic properties and phylogenetic relationship.

MATERIALS AND METHODS Virus strains and RNA preparation

*Corresponding author: Dr Hung-Jen Liu, Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan, ROC. Fax: +886-8-7740205; E-mail: [email protected] 0034-5288/01/020139 + 09 $35.00/0

Five IBDV isolates, including V97/TW, T1/TW, 2512, T2/CH and Lukert, were used in this study. Two isolates, V97/TW and T1/TW, were field-isolates from Taiwan and T2/CH from © 2001 Harcourt Publishers Ltd

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China. 2512 strain of IBDV was a vaccine strain used in Taiwan. Lukert strain of IBDV was a classical vaccine strain from the USA. To prepare a cloned cDNA probe used in ISH, Lukert strain was propagated in primary chicken embryo fibroblast (CEF) cells. Upon development of 70–80 per cent cytopathic effect (CPE), the cell cultures were frozen and stored at –70˚C until used. In the case of the other isolates, infected birds were killed and the bursa of Fabricius removed. The bursae were immediately placed in TNE buffer [10 mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA]. Cells were harvested from a crude suspension prepared by homogenising the entire bursae in buffer. The cell suspensions were then frozen at –70˚C until needed. Viral particles were purified following methods described previously (Liu et al 1994). Briefly, viral particles were concentrated by polyethylene glycol-6000, pelleted and resuspended in TNE buffer (0.01 M Tris-HCl, pH 7.6; 0.1 M NaCl and 0.001 M EDTA), followed by extraction with Freon (1,1,2-trichlorotrifluoroethane; Sigma Co., St. Louis, MO, USA). The supernatants containing virions were centrifuged at 50 000 × g for 2 hours at 4˚C through a 35 per cent sucrose cushion, and the viral pellets obtained were then centrifuged isopycnically in stepwise gradients 40, 30 and 20 per cent (w/w) CsCl. The virus particles banding at a buoyant density of 1.34 g ml–1 were withdrawn and concentrated by centrifugation at 132 000 × g for 2 hours at 4˚C. Purified virus particles were resuspended in TNE buffer containing 0.5 per cent sodium dodecyl sulfate (SDS). Proteinase K was added to a final concentration of 1 mg ml–1. After incubation for 1 hour at 37˚C, the viral RNA was isolated by two consecutive phenol:chloroform:isoamyl alcohol (25:24:1) extractions and recovered by precipitation with 100 per cent ethanol containing 0.4 M LiCl. The dsRNA was further purified by LiCl fractionation precipitation (Diaz-Ruiz and Kaper 1978). The viral RNA was washed twice with 70 per cent ethanol to remove LiCl and suspended in diethylpyrocarbonate (DEPC)treated water. The purified viral RNA was stored at –70˚C until used. Experimental inoculation of chickens One hundred and five 3-week-old specific-pathogenicfree (SPF) White Leghorn chicks were used. The chicks were equally divided into seven groups. Group I to group VI chicks were given the virus strains V97/TW, T1/TW, 2512, Lukert, PBG98, and D78, respectively. Group VII chicks were unchallenged (control). Each bird was inoculated in one nostril and one eye, each, with 50 µl of a virus suspension containing at least 10 chicken infected doses per ml. The birds were housed in separate modified Horsfall-Bauer isolation units maintained with filtered air. Three days postinoculation chick tissues were collected for ISH study and pathological examination. Reverse transcription and polymerase chain reaction (RT-PCR) To identify IBDV in the infected bursa of Fabricius, the was extracted and then amplified by RT-PCR. Two sets of primer pairs (P1-P2 and P3E-P4E) were used and described previously (Liu et al 1994, Liu 2000). Primer pairs P1-P2 and P3E-P4E were chosen according to the cDNA sequences of IBDV strain Cu-1 genome segment A

IBDV RNA

(Spies et al 1987). The sequence of primers is as follows: P1 primer, 5′-TCACCGTCCTCAGC TTAC-3′ (identical to nucleotides 622 to 639), and the P2 primer, 5′-TCAGGATTTGGGATCAGC-3′ (complementary to nucleotides 1264 to 1247); primer P3E, 5′-TTAGAATTCATCAGACAAACG-3′ (identical to nucleotides 101 to 121) and P4E, 5′-TTCCGAATTCTGTCGTTGATG-3′ (complementary to nucleotides 580 to 600). To efficiently clone the PCR product, PCR primers P3E and P4E with an EcoRI restriction site (underlined) were designed. The amplified cDNA fragment using primer pairs P1-P2 and P3E-P4E is expected to be 643 bp and 500 bp long, respectively. PCR was performed according to procedures provided by Perkin Elemer Co. (Branchburg, NJ, USA). The reverse transcription was carried out at 60˚C for 30 minutes. PCR reaction was carried out in a total volume of 100 µl containing 1x EZ buffer, 2.5 mM manganese acetate solution, 300 µM dNTPs, rTth DNA polymerase (10 units), IBDV RNA (1 µg) and 5 µM of the primers (P1-P2 or P3E-P4E). PCR reactions were subjected to 35 cycles consisting of denaturation for 1 minute at 94˚C, annealing for 2 minutes at 60˚C and one final extension cycle at 60˚C for 7 minutes. After completion of the PCR, 5 µl of the reaction mixture was loaded onto a 1.2 per cent agarose gel containing 0.5 µg ml–1 ethidium bromide for electrophoresis and subsequent visualisation by UV transillumination. Preparation of digoxigenin (DIG)-labelled probe To prepare a cloned cDNA probe used in ISH, a set of primer (P3E and P4E) chosen from the 5′ domain of the genome segment A of IBDV, was designed to amplify a conserved region of the VP2 gene of IBDV. PCR product (500 bp) amplified from IBDV Lukert strain was purified according to Magic™ PCR Preps DNA Purification Kit procedure (Promega Co., Madison, WI, USA). Purified PCR product was digested with restriction enzyme EcoRI. The digestion was incubated for 2 hours at 37˚C. The EcoRI-digested cDNA was cloned into the EcoRI site of dephosphorylated plasmid pUC 18, then transformed into Escherichia coli competent cells (Sambrook et al 1989). The white colonies carrying recombinant plasmids were selected from Luria-Bertani (LB) agar plates containing ampicillin (50 µg ml–1) and 25 µl of 40 mg ml–1 X-gal stock solution. The alkaline lysis method was used for small preparations of plasmid DNA. DNA labelling was performed according to the manufacturer’s procedure (Boehringer Mannheim). In situ hybridisation Chickens infected by V97/TW strain of vvIBDV were killed and infected tissues removed for ISH and pathology examination. Chicken tissues, including bursae, spleen and intestine were collected. A small fragment from each organ was fixed in 10 per cent neutral buffered formalin for 20 hours. The tissues were embedded in paraffin. The formalin-fixed, paraffin-embedded tissues were sectioned at 5 µm thick and placed on saline-coated slides. Sections stained with haematoxylin and eosin (H&E) were evaluated for lesions. The diagnosis was based upon microscopic lesions of H&Estained sections. Unstained sections were used in ISH assays. Pretreatment of sections for ISH was done as described previously (Liu et al 1997). The ISH solution contained 50 per

Infectious bursal disease viruses in Taiwan

A bp

1

2

3

4

B.

5

2

3

4

bp

1000 700

1

643

141

C

5 bp

600 500 400

bp 2690

2

1 3000 2000

500 500

600

500

FIG 1: Detection of infectious bursal disease virus (IBDV) by using reverse transcription polymerase chain reaction (RT-PCR). (A) Agarose gel electrophoresis of PCR-amplified cDNA fragment (643 bp). Lane 1, Bio100 DNA ladder™ molecular weight marker; lane 2, T2/China; lane 3, V-97/TW; lane 4, 2512; lane 5, T1/TW. (B) Agarose gel electrophoresis of PCR-amplified cDNA fragment (500 bp). Lane 1, molecular weight markers (50–1000 bp); lane 2, T2/China; lane 3, V-97/TW; lane 4, 2512; lane 5, T1/TW. (C) Cloning of PCR-amplified cDNA fragment (500 bp). Lane 1, Bio100 DNA ladder™ molecular weight marker; lane 2, Lukert strain

cent formamide, 5x SSC, 2 per cent blocking reagent, 1 per cent N-lauroylsacosine, 0.02 per cent SDS, and DIG-labelled cDNA probe at a concentration of 3 µg ml–1. Prehybridisation was performed without the DIG-labelled cDNA probe for 1 hour at 42˚C. Prior to use, the hybridisation solution was boiled for 10 minutes and then cooled in ice to denature the probe. 15 µl of the cocktail was pipetted onto each section. Each section was covered with a glass coverslip and sealed with paraffin. The sections were placed in a moist chamber and hybridised at 42˚C overnight. Following hybridisation, the sections were washed twice in 2x SSC/ 0.1 per cent SDS for 15 minutes at room temperature and twice in 0.1 per cent SSC/ 0.1 per cent SDS for 15 minutes at 68˚C. Immunological detection was performed according to the manufacturer’s procedure (Boehringer Mannheim). The sections were washed in washing buffer (100 mM Tris-HCl buffer, pH 7.5; 150 mM NaCl). A blocking reagent was applied to the sections for 30 minutes at room temperature. After washing for 2 minutes in the washing buffer, the sections were incubated for 30 minutes at room temperature with sheep anti-DIG antibodies conjugated to alkaline phosphatase, and diluted 1:10000 in washing buffer. Unbound anti-DIG antibodies were removed by washing slide sections twice in washing buffer for 15 minutes. The sections were equilibrated for 3 minutes with equilibration buffer (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.5). The alkaline phosphatase substrate, including 45 µl of nitroblue tetrazolium (NBT) solution and 35 µl of X-phosphate solution, were added to 10 ml of equilibration buffer. Development of dark brown colour reaction proceeded until positive signals were observed under a microscope. The colour development was stopped by washing sections in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) for 5 minutes. Controls included ISH or uninfected tissues with or without the IBDV cDNA probe. Controls were processed in an identical manner.

alkaline lysis method were performed on white colonies containing the VP2 gene were cleaved with EcoRI/ Hind III. Recombinant plasmid DNA was purified using a QIAGEN purification Kit (QIAGEN, Valencia, CA, USA) and sequenced with an automated Laser Fluorescence DNA Sequencer. Sequence alignment and phylogenetic analysis Nucleotide and deduced amino acid sequences of the HVR of VP2 were aligned and analysed with a Clustal method of DNASTAR software package (DNASTAR Inc., Madison, WI, USA). The nucleotide sequences (435 bp) of the VP2 gene were aligned and compared, corresponding to residues 206–350 of the VP2 protein. The cDNA sequences and predicted amino acid sequences of the VP2 protein were used to determine the extent of genetic and antigenic diversity. Nucleotide sequences (435 bp) from the HRV of VP2 were used to generate a phylogenetic tree for relationship study. In the present study, the HVR of the VP2 gene corresponding to amino acid residues 206–350 was used to create a phylogenetic tree. The nucleotide sequences data reported in this paper have been submitted to the GeneBank nucleotide database and have been assigned accession numbers: AF279288 (2512), AF279287 (V97/TW), AF303219 (T1/TW), AF312371 (T2/China). GeneBank accession numbers and isolation place for other sequences used in phylogenetic analysis were as follows: AF109154 (P3009, Taiwan), AF006697 (GZ911, China), AF092171 (Harbin, China), AF121256 (Hangzhou HZ296, China), AF006699 (GZ902, China), AF006695 (F9502, China), D49706 (OKYM, Japan), TABLE 1: The mortality in each group of chicks challenged with vvIBDV, attenuated IBDV and classical virulent strains Days after post challenge

Cloning and sequencing of cDNA To investigate genetic variation and phylogenetic relationships of IBDV, the HVR of the VP2 gene of IBDV were cloned and sequenced. Purified PCR products (643 bp) derived from the VP2 gene were treated with klenow polymerase and T4 polynucleotide kinase and then inserted into the Sma I site of dephosphorylated plasmid pUC18 (Sambrook et al 1989). Recombinant plasmids were used to transform E coli competent cells. DNA minipreps using

Group I II III IV V VI VII

Virus strains V97/TW T1/TW 2512 Lukert PBG98 D78 Control†

3 days 15* 10 0 0 0 0 0

7 days

10 days

0 2 1 0 0 0 0

0 0 0 0 0 0 0

*Number of dead chicks; † Chicks given with PBS buffer.

Mortality (percentage) 100 80 6.6 0 0 0 0

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AF148076 AF140705

(01/94, Australia), AF148081 (08/95, Australia), (Soroa, Spain), X84034 (P2, Germany), D00867 (Cu-1, Germany), AF159216 (K357/88, Germany), X95883 (849VB, Belgium), D00868 (PBG98, Great Britain), D00869 (52/70, Great Britain), X92760 (UK661, Great Britain), AF159215 (N14, Nigeria), AF159218 (K406/89, Egypt), AF159217 (K280/89, South Africa), AF091097 (3212, USA), AF091099 (U28, USA), Y14962 (D78-Lab, USA), M64285 (Variant A, USA), D10065 (Variant E, USA), M97346 (GLS, USA), AF091098 (MISS, USA), AF076236 (Univax, USA), D00499 (STC, USA), D16679 (Lukert, USA), AJ238647 (MYGA-97, Cuba).

RESULTS RT-PCR

and CDNA coloning

The dsRNA from four IBDV isolates was reversetranscribed to cDNA and amplified. All amplified cDNA showed almost identical mobilities on a 1.2 per cent agarose gel. PCR amplification using primer pairs P1-P2 and P3EP4E generated a specific DNA band of 643 bp and 500 bp, respectively, as expected (Fig 1A, B). RT-PCR detected IBDV in bursae and further confirmed the specificity of ISH. To prepare a cloned cDNA probe used in ISH, a cDNA fragment (500 bp) derived from the 5′-domain of the VP2 gene was cloned into pUC18 plasmid (Fig 1C). Pathological examination of experimentally infected chickens The vvIBDV field isolates produced severe clinical signs and high mortality in chickens, causing over 70–100 per cent mortality in chickens in Taiwan. Experimentally IBDVinfected SPF chicks were examined at 3, 7 and 10 days postchallenge (PC). The very virulent field isolates produced widespread necrosis of lymphocytes and lymphoid follicles and cystic cavities in medullary areas of follicles. The Taiwanese vvIBDV strains caused up to 80–100 per cent mortality in SPF chicks (Table 1), suggesting that they were very virulent viruses. In situ detection of vvIBDV in chicken tissues By using ISH technique, the IBDV genome was detected in bursae and spleen tissues. The greatest number of positive signals were observed in bursae (Fig 2B, C). Staining was mainly observed in the cortex and medullary areas of follicles and interfollicle tissues. Only a few positive signals were observed in spleen (Fig 2A). No staining was observed in sections from intestine and negative controls. ISH results were consistent with lesions observed in bursae and spleen. FIG 2: In situ hybridisation (ISH) for the detection of infectious bursal disease virus (IBDV) in (A) spleen and bursae (B, C). Slides reacted with a digoxigenin (DIG) labelled IBDV probe. Positive signals were detected in spleen and bursal tissues. Arrows indicated the examples of the positive signals. Magnification: A (400 x), B (400 x), C (100 x)

AB024076 AJ249523

(Ehime 91, Japan), AFD16828, (GBF-1, Japan), (Tri-bio, India), AJ249519 (Vaclnetermediate, India), L42284 (KS, Israeli), X03993 (002-73, Australia),

Sequence analysis The nucleotide sequences of the HVR of VP2 located between nucleotide 618 and 1050 were determined for three IBDV isolates and one vaccine strain 2512. To elucidate how the recent vvIBDV appeared in Taiwan, the deduced amino acid sequences of the HVR of VP2 (residues 206–350) of

Infectious bursal disease viruses in Taiwan

143

FIG 3: Alignment and comparison of deduced amino acid sequences of hypervariable region (HVR) in the VP2 gene of various serologic standard and variant isolates. The sequence of isolate V97/TW is shown on top, and only differences are indicated. The amino acid sequence is listed between enzyme sites AccI and SpeI which includes residues number 206 to 350. A conserved region in the virulent isolates is underlined. The two large boxed in areas included the hydrophilic regions responsible for the neutralising epitopes

isolate V97/TW were compared with those of the classical virulent, variant, and vvIBDVs (Fig 3). Three amino acids were identified as specific for isolate V97/TW at amino acid positions 208, 228 and 266. The serine-rich heptapeptide326 SWSASGS332 next to the second hydrophilic region was conserved in recent Taiwanese isolates, and two amino acid substitutions at positions 330 (S to M) and 331 (G to W) were found in the Taiwanese classical P3009 strain. Phylogenetic relationship of Taiwanese strains to other IBDV strains A phylogenetic tree based upon nucleotide sequences of the HVR of VP2 was constructed showing the relationship of

Taiwanese strains to other IBDV strains (Fig 4). The IBDV strains were classified into four groups: (i-1 & 2) classical and variant IBDV strains formed one large group; (ii) vvIBDV formed a separate group not too distantly related to group (i); (iii) Australian variants; (iv) Australian classical strain. Phylogenetic analysis showed that Taiwanese IBDV isolates belong to two distinct genetic groups which are considerably heterogeneous. Recent Taiwanese isolates T1/TW and V97/TW were similar to vvIBDV from other countries. P3009 strain of IBDV was phylogenetically related to the classical vaccine Lukert-BP strain.

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FIG. 3 (cont)

DISCUSSION New virulent strains of IBDV have appeared all over the world in the last few years (Lin et al 1993, Brown and Skinner 1996, Yamaguchi et al 1996, Pitcovski et al 1998). In Taiwan, the vvIBDV strains have caused high mortality rates in chickens. The vvIBDV RNA could be readily detected and localised in the bursa of Fabricius and spleen of experimently infected chickens and IBDV-infected field samples. When ISH combined with a cloned DIG-labelled cDNA probe, ISH could be done within 24 hours. Therefore, ISH could be utilised as a safe, rapid and accurate method for examining large numbers of field samples or as a rapid screening for selected tissues. Alternation in IBDV features have been reported in several countries (Lin et al 1993, Brown and Skinner, 1996, Yamaguchi et al 1996, Pitcovski et al 1998, Sapata and

Ignjatavic 2000). In the US and Australia, some isolates were shown to change their antigenic properties as defined by using monoclonal antibodies (Snyder et al 1992, Sapata and Ignjatavic 2000). The vvIBDV strains were antigenically very similar to classical IBDV strains but were able to break through the usually protective levels of maternal antibodies induced by mild and classical vaccines (Van den Berg and Meulemans 1991, Godard et al 1994). In recent years, these vvIBDV strains have been shown to be undergoing antigenic change (Eterradossi et al 1998). As reports described previously, sequence comparisons of the HVR of VP2 among strains can offer the evolutionary clues for IBDV antigenic and virulent characteristics. The boxed hydrophilic regions (Fig 3) located at the HVR of VP2 gene have been shown to be important in the binding of neutralising monoclonal antibodies and responsible for serotype specificity (Azad et al 1987, Becht et al 1988, Fahey et al 1989). Molecular data

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145

FIG. 3 (cont)

from the recent vvIBDV and classical strains from Taiwan revealed interesting results. The sequences of the recent Taiwanese isolates are closely related to those of the vv strains from Europe, China, Japan and Africa. Two amino acid substitutions at positions 217 (L to S) and 222 (P to A), existing in recent Taiwanese vvIBDV strains, might induce antigenic differences that could cause the failure of the classical vaccine strain 2512 to induce full protection against the vvIBDV in Taiwan (Vakharia et al 1994). It is interesting to note that three amino acids are unique to isolate V97/TW at amino acid positions 208L, 228I, and 266L, compared with all reported IBDV strains. Aligning the HVR of all reported

vvIBDV strains makes it obvious that three of unique amino acid residue were identified as specific for the vv strains at positions 222 (A), 256 (I), and 294 (I). These amino acids were also conserved in the recent Taiwanese strains. However, the conservation of amino acid may play an important role in increased virulence of vv strains. The serine rich heptapeptide 326SWSASGS332 located immediately downstream of the second hydrophilic region was believed to be involved in the virulence of IBDV (Heine et al 1991, Vakharia et al 1994) and was conserved in the recent Taiwanese isolates, indicating that they were pathogenic strains. In contrast, two amino acid substitutions at positions

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Cu-1 D78-Lab T2/China Tri-bio PBG98 Soroa P2 GZ911 Harbin Univax Hangzhou HZ96 Vacintermediate

(i-1)

GZ902

Classical and variant starins

Variant A 3212 Variant E U28 GLS GBF-1 52/70 STC 2512 G9201 HK46 K357/88 K406/89 Ehime 91 OKYM XJ-9 MYGA-97 K280/89

(ii)

vvIBDV

UK661 KS V97/TW T1/TW N14 849VB P3009/TW Lukert-BP

(i-2)

Classical and variant starins

(iii)

Australian variant

F9502 MISS 08/95 01/94 002-73 10.5 10

8

6

4

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Australian classica strain

0

FIG 4: Phylogenetic tree based on nucleotide sequence located in the hypervariable region (HVR) of the VP2 gene of various serologic standard and variant isolates using the Clustal program of the DNASTAR software package

Infectious bursal disease viruses in Taiwan

330 and 331 were found in the Taiwanese classical strain P3009, suggesting that it was a less virulent strain having fewer serine residues (Heine et al 1991, Vakharia et al 1994). The other two amino acids at positions 279D and 284A were found to be conserved in the vvIBDV strains, suggesting that the amino acids at these positions might contribute to virulence of IBDV. A denogram of the alignment of the HVR on the VP2 gene showed four distant groups among IBDV isolates. Phylogenetic analysis in this study showed that the Taiwanese vv strains clustered with European, Japanese, Chinese and African vvIBDV strains in the phylogenetic tree regardless of geographic locations of their isolation. Sequence and phylogenetic analysis revealed that the recent Taiwanese strains evolved closely and separately from classic virulent and vaccine strains. Data reported herein on the HVR of VP2 sequences of recent Taiwanese IBDV isolates provided valuable information on the evolution of these isolates and the molecular mechanism for antigenic and pathogenic changes. ACKNOWLEDGEMENTS This research work was supported by the Council of Agriculture, Taiwan. REFERENCES AZAD, A. A., JAGADISH, M. N., BROWN, M. A. & HUDSON, P. J. (1987) Deletion mapping and expression in Escherichia coli of the large genomic segment of a birnavirus. Virology 161, 145–152 BAGASRA, O., HAUPTMAN, S. P. & LISCHER, H. W. (1992) Detection of human immunodeficiency virus type 1 provirus in mononuclear cells by in situ polymerase chain reaction. New England Journal of Medicine 326, 1385–1391 BAYLISIS, C. D., SPIES, U., SHAW, K., PETERS, R. W., PAPAGEORGIOU, A., MULLER, H. & BOURSNELL, M. E. G. (1990) A comparison of the sequences of segment A of four infectious bursal disease virus strains and identification of a variable region in VP2. Journal of General Virology 71, 1303–1312 BECHT, H., MULLER, H. & MULLER, H. K. (1988) Comparative studies on structural and antigenic properties of two serotypes of infectious bursal disease virus. Journal of General Virology 69, 631–640 BROWN, M. D. & SKINNER, M. A. (1996) Coding sequences of both genome segments of a European very virulent infectious bursal disease virus. Virus Research 40, 1–15 CAO, Y. C., YEUNG, W. S., LAW, M., BI, Y. Z., LEUNG, F. C. & LIM, B. L. (1998) Molecular characterization of seven Chinese isolates of infectious bursal disease virus: classical, very virulent, and variant strains. Avian Disease 42, 340–351 CHETTLE, N. J., STUART, J. C. & WYETH, P. J. (1989) Outbreaks of virulent infectious bursal disease in East Anglia. Veterinary Record 125, 271–272 CUMMINGS, T. S., BROUSSARD, C. T., PAGE, R. K., THAYER, S. G. & LUKERT, P. D. (1986) Infectious bursal disease virus in turkeys. Veterinary Bulletin 56, 757–762 DIAZ-RUIZ, J. R. & KAPER, J. M. (1978) Isolation of viral double-stranded RNAs using a LiCl fractionation procedure. Preparative Biochemistry and Biotechnology 8, 1–17 ETERRADOSSI, N., ARNAULD, C., TOQUIN, D. & RIVALLAN, G. (1998) Critical amino acid changes in VP2 variable domain are associated with typical and atypical antigenicity in very virulent infectious bursal disease viruses. Archives of Virology 70, 1473–1481 FAHEY, K. J., ERNY, K. & CROOKS, J. (1989) A conformational immunogen on VP2 of infectious bursal disease virus that induces virus-neutralizing antibodies that passively protect chickens. Journal of General Virology 70, 1473–1481 GODARD, R. D., WYETH, P. J. & VARNEY, W. C. (1994) Vaccination of commercial layer chicks against infectious bursal disease with maternally derived antibodies. Veterinary Record 17, 237–274 HEINE, H. G., HARITOU, M., FAILLA, P., FAHEY, K & AZAD, A. (1991) Sequence analysis and expression of the host-protective immunogen VP2 of a variant strain of infectious bursal disease virus which can circumvent vaccination with standard type I strains. Journal of Virology 72, 1835–1843 HUDSON, P. J., MCKERN, N. M., POWER, B. E. & AZAD, A. A. (1986) Genomic structure of the large RNA segment of infectious bursal disease virus. Nucleic Acids Research 14, 5001–5012

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