Journal of General Virology (1990), 71, 569-577. Printed in Great Britain

569

Nucleotide sequence analysis of genome segment A of infectious bursal disease virus Frederick S. B. Kibenge,*1" Daral J. Jackwood and Cynthia C. Mercado Food and Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 44691-4096, U.S.A.

The nucleotide sequence of genome segment A cDNA of the STC strain of infectious bursal disease virus (IBDV) was determined and compared with sequences of the homologous genome segment of the 002-73 strain of IBDV and the Jasper strain of infectious pancreatic necrosis virus (IPNV). The STC-IBDV genome segment A was determined to be 3262 base pairs (bp), which is close to the estimated total length of 3300 bp for genome segment A in IBDV, although there is no proof that it is the real length of this genome segment. The STC-IBDV genome segment A contains two major overlapping open reading frames (ORFs). The large O R F of 3036 bp predicts a polyprotein of Mr 109 358, whereas the small O R F is 435 bp and predicts a protein of Mr 16550 in STC-IBDV. STC-IBDV and 002-73-IBDV polyproteins are closely related (97.4% amino acid homology). Most of the amino acid

mismatches are in VP2 sequences, mainly within the area of the conformation-dependent epitope. Comparison with the Jasper-IPNV polyprotein reveals levels of amino acid sequence homology of about 40 % in VP2, 32% in VP3 and 21% in VP4. Within the VP2 molecule the conformation-dependent epitope area is again the least homologous, but the heterogeneity is more conspicuous than between the two IBDV strains, which is not surprising since IBDV and IPNV are serologically unrelated. The small O R F proteins have about 88% amino acid sequence homology between STC-IBDV and 002-73-IBDV, and 30 % between each IBDV strain and Jasper-IPNV. There is no homology at all in the non-coding regions of IBDV and IPNV. These comparative sequence data will be useful for subgrouping the Birnaviridae family.

Introduction

IBDV is a member of the birnavirus genus, family Birnaviridae (Brown, 1986) and infectious pancreatic necrosis virus (IPNV) of fish is the prototype of the family. The genome of these viruses shows homology in organization and function and consists of two dsRNA molecules, designated segments A and B (Macdonald & Dobos, 1981). Genome segment B (approx. 2800 bp) encodes VP1, the putative dsRNA polymerase (Morgan et al., 1988; Muller & Nitschke, 1987a, b; Spies et al., 1987), whereas the larger segment A (approx. 3300 bp) encodes a precursor polyprotein that is processed into mature VP2, VP3 and VP4 (Hudson et al., 1986; Muller & Nitschke, 1987b). VP2 and VP3 are the major structural proteins of the virion; VP2 contains the antigenic regions responsible for inducing neutralizing antibodes and for serotype specificity (Azad et al., 1987; Becht et al., 1988; Fahey et al., 1989), whereas VP3 contains the group-specific antigens (Becht et al., 1988). Deletion expression studies of the cDNA fragment of genome segment A of 002-73-IBDV (Azad et al., 1987;

Infectious bursal disease virus (IBDV) is a pathogen of considerable economic interest to the poultry industry world-wide. Two distinct serotypes oflBDV (1 and 2) are recognized. Serotype 1 virus strains differ markedly in virulence (Winterfield & Thacker, 1978), whereas all known serotype 2 viruses are naturally avirulent for chickens (Ismail et al., 1988). An explanation at the molecular level for the biological differences among IBDV isolates is lacking. Such information would aid in development of better diagnostic tools and in the construction of ideal vaccines for protection against infectious bursal disease in chickens (Kibenge et al., 1988). t Present address: Department of Pathology and Microbiology, Atlantic VeterinaryCollege, Universityof Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island C1A 4P3, Canada.

0000-9195 © 1990 SGM

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F. S. B. Kibenge, D. J. J a c k w o o d and C. C. M e r c a a o

J a g a d i s h et al., 1988) a n d o f J a s p e r - I P N V ( D u n c a n et al., 1987) i n d i c a t e d t h a t V P 4 is i n v o l v e d in the p r o c e s s i n g o f the p r e c u r s o r p o l y p r o t e i n , suggesting t h a t V P 4 m a y be a v i r u s - e n c o d e d protease. T h e e x a c t locations o f the c l e a v a g e sites w i t h i n the p o l y p r o t e i n are not k n o w n a n d the VP4 c o d i n g regions o f 002-73-IBDV a n d J a s p e r I P N V s h o w e d no significant sequence h o m o l o g y ( A z a d et al., 1987; J a g a d i s h et al., 1988). c D N A s e q u e n c i n g studies h a v e shown t h a t g e n o m e s e g m e n t A o f J a s p e r - I P N V has two o p e n r e a d i n g f r a m e s ( O R F s ) . A large O R F o f 2916 b p e n c o d i n g the p r e c u r s o r p o l y p r o t e i n has 5' a n d 3' n o n - c o d i n g regions o f 119 a n d 62 nucleotides, respectively ( D u n c a n & D o b o s , 1986). T h e 5' n o n - c o d i n g region c o n t a i n s an i n i t i a t o r c o d o n at nucleotide p o s i t i o n 68 for a small O R F o f 444 bp, w h i c h o v e r l a p s w i t h the 5' e n d o f the p o l y p r o t e i n O R F a n d could e n c o d e a 17K p o l y p e p t i d e ( D u n c a n et al., 1987). I n 002-73-IBDV, the only o t h e r b i r n a v i r u s whose g e n o m e has been sequenced, the p o l y p r o t e i n O R F o f 3036 b p w i t h 5" a n d 3' t e r m i n a l f l a n k i n g regions o f 28 a n d 65 nucleotides, respectively, has b e e n r e p o r t e d ( H u d s o n et al., 1986). N o o t h e r O R F s h a v e b e e n r e p o r t e d for g e n o m e s e g m e n t A o f this virus. Since no c o m p a r a t i v e sequence d a t a are a v a i l a b l e for the g e n o m e s o f e i t h e r I B D V or I P N V , the n a t u r e o f the g e n o m e s e g m e n t A v a r i a t i o n b e t w e e n these B i r n a v i r i d a e m e m b e r s is not known. In an a t t e m p t to u n d e r s t a n d b e t t e r the m o l e c u l a r basis for the biological differences a m o n g I B D V isolates and the r e l a t i o n s h i p o f I B D V w i t h o t h e r birnaviruses, the p r i m a r y sequences o f viral g e n o m e s f r o m selected I B D V strains are b e i n g d e t e r m i n e d . W e r e p o r t here the nucleotide a n d d e d u c e d a m i n o a c i d sequences o f g e n o m e s e g m e n t A o f the highly p a t h o g e n i c A m e r i c a n S T C I B D V . Sequence c o m p a r i s o n s o f this serotype 1 virus were m a d e w i t h p u b l i s h e d sequences o f the h o m o l o g o u s g e n o m e s e g m e n t o f the A u s t r a l i a n 002-73-IBDV (a serotype 1 virus o f low p a t h o g e n i c i t y ) a n d J a s p e r - I P N V (a serologically u n r e l a t e d m e m b e r o f the b i r n a v i r u s genus). W e also r e p o r t a d d i t i o n a l nucleotide sequences at b o t h the 5' a n d 3' t e r m i n i o f g e n o m e s e g m e n t A o f I B D V a n d d e s c r i b e the a p p a r e n t basic g e n o m i c o r g a n i z a t i o n o f I B D V , w h i c h m a y be the s a m e for o t h e r b i r n a v i r u s e s .

Synthesis and cloning of STC-IBD V cDNA. STC-IBDV was propagated in 3-week-old, specific pathogen-free chickens. The viral RNA was isolated and eDNA was prepared as previously described (Jackwood et al., 1989). Briefly, random primers made from calf thymus DNA were used to prime the reverse transcription of purified viral RNA in the first-strand reaction. Second-strand synthesis was carried out as described by Gubler & Hoffman (1983). STC-IBDV double-stranded cDNA was annealed into the oligo(dG)-tailed PstI site of pUC9 and used to transform competent Escherichia coli JM107 cells. Techniques for identification of IBDV-specific eDNA clones were as described (Jackwood et al., 1989), with the following modifications. The cDNAs that hybridized to genome segment A of STC-IBDV in Northern blots were used in colony blot hybridization (Grunstein & Hogness, 1975) to identify related cDNAs within the library. These viral clones were analysed for plasmid size on 1% agarose gels and selected clones were prepared for sequence analysis. This procedure was repeated until individual viral clones with overlapping eDNA inserts that spanned the whole coding region of genome segment A STC-IBDV were identified. Isolation ofplasmid DNA. Both the rapid boiling method of Holmes & Quigley (1981) and the alkaline lysis method of Birnboim & Doly (1979) were used for small preparations (mini-preps) for plasmid DNA. The alkaline lysis method of Birnboim (1983) was used for larger preparations of plasmid DNA, except that plasmids were purified either by fractionation on Bio-Gel A-50 columns (Bio-Rad) following phenol--chloroform-isoamyl alcohol extraction and ethanol precipitation (Maniatis et al., 1982; Raymond et al., 1988), or by precipitation from polyethylene glycol (PEG) (0.4 vol. of 30% PEG 8000 in 1.8 MNaC1 at 4 °C overnight). PEG was removed by chloroform isoamyl alcohol extraction and plasmid DNA was precipitated with either cold ethanol or isopropanol. Sequencing of lBDV eDNA clones and sequence analysis. Denatured double-stranded plasmid DN A was sequenced by a modification of the dideoxynucleotide chain termination procedure (Chen & Seeburg, 1985) with either the M13/pUC universal or reverse primer (Boehringer) and [ct-35S]ATP (sp. act. >600 Ci/mmol; Amersham). The Klenow fragment of DNA polymerase I (U.S. Biochemical Corporation), the modified bacteriophage T7 DNA polymerase (Sequenase; U.S. Biochemical Corporation) or the genetically engineered form of Sequenase (Sequenase version 2-0) were used in the sequencing reactions. Six percent polyacrylamide-8 M-urea electric field gradient gels (Ansorge & Labeit, 1984) in 100 mM-TrispH 8.3, 83 mM-boricacid and 1 mM-EDTA were used to resolve the reaction products. Nucleotide sequence data were compiled and analysed on an IBM Personal Computer XT using the Bionet software system (Intelligenetics Incorporated) and the FASTA program package for microcomputers (Pearson & Lipman, 1988).

Results Methods

Determination o f S T C - I B D V genome segment A nucleic acid sequences

Virus. The virus used for this study was infectious bursal disease standard challenge (STC) virus, as distributed by the United States Department of Agriculture. This virus (designated here as STC-IBDV) was originally isolated from chickens by Dr S. A. Edgar (Cheville, 1967).

T r a n s f o r m a t i o n o f c o m p e t e n t E. coli JM107 cells w i t h the r e c o m b i n a n t p l a s m i d s yielded 271 white colonies on L u r i a agar plates c o n t a i n i n g 100 p.g a m p i c i l l i n p e r ml, 0-1 m M - I P T G a n d 0.002% X - G a l . By c o l o n y b l o t h y b r i d i z a -

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Fig. 1. Primary nucleotide sequence of the plus strand of STC-IBDV genome segment A cloned cDNA. The deduced amino acid sequences (in one-letter code) for the small ORF and the polyprotein (large) ORF are centred over their respective codons. The last nucleotide or amino acid in each line is numbered at the right. The small ORF extends from nucleotide positions 63 to 497 and the polyprotein ORF from 97 to 3132. The initiation and stop codons are underlined and the stop codons are also indicated by asterisks. The proposed cleavage sites (KR, RR and AXAAS residues) for the IBDV polyprotein (Azad et al., 1987; Jagadish et al., 1988) are overlined. The area within vertical arrows represents the conformation-dependent epitope recognized by monoclonal antibody 17/82 (Azad et al., 1987). Amino acids below the major sequences are substitutions found in 002-73-IBDV (Hudson et al., 1986); non-conservative changes are printed in bold and double underlined. Amino acid homology with Jasper-IPNV is underlined. The inverted and complementary terminal repeat sequences are boxed. Bracketing arrows indicate regions with neither nucleotide nor amino acid sequence homology between STC-IBDV and Jasper-IPNV:

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R Q Y H L A M A A S E F K ~ T P E L E S A V R A 771 A C G C C A H T A C C A C C T T G C C A T G G C T G C A T C A G A G T T C A A A G A G A C C C C T G A A C T C G A G A G C G C C G T C A G A G C C 2409 M E A A A N V D P L F Q S A L S V F M W L E E N 795 A T G G A A G C A G C A G C C A A C G T G G A C C C A C T A T T C C ~ A T C G G C A C T C A G T G T G T T C A T G T G G C T G G A A G A G A A T G 2482 D G I V T D M A N F A L S D p N A H R M R N F L A N 82O G G A T T G T G A C C G A C A T G G C C A A C T T C G C A C T C A G C G A C C C G A A C G C C C A T C G G A T G C G A A A T T T T C T T G C A A A 2555

tion analysis and plasmid D N A sequence analysis at least 104 r e c o m b i n a n t plasmids were confirmed to have overlapping c D N A inserts, ranging from 43 to 643 bp, that covered the entire coding region o f g e n o m e s e g m e n t A o f S T C - I B D V . T h e nucleotide sequence o f 3262 bp o f S T C - I B D V g e n o m e s e g m e n t A (Fig. 1) was c o m p i l e d from 17 c D N A inserts that aligned with sequences o f the homologous genome segment of 002-73-IBDV (Hudson et al., 1986). W i t h the exception o f the extreme 5' and 3' ends this sequence was obtained throughout from at least two i n d e p e n d e n t overlapping inserts by s e q u e n c i n g both strands o f the D N A . W h e n the s e q u e n c e differed b e t w e e n two clones a third and occasionally a fourth clone was sequenced in this area. T h e extreme 5' and 3'

572

F. S. B. Kibenge, D. J. Jackwood and C. C. Mercado

sequenCes were obtained from single clones and were not confirmed by direct sequencing on the genomic RNA. Therefore, we do not know whether the 3262 bp is the real length of this genome segment.

IBDV Polyprotein 5 ' ........

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30

G*C G*C

Evidence for two major OREs in genome segment A of IBDV The plus strand of genome segment A of STC-IBDV contains two major overlapping ORFs, positioned in different reading frames (Fig. 1 and 3). The large continuous ORF of 3036 bp, as in the 002-73-IBDV c D N A sequence (Hudson et al., 1986), encodes a 1012 amino acid precursor polyprotein. The predicted Mr of the STC-IBDV polyprotein is 109358, whereas that of 002-73-IBDV is 109413. In contrast the translation product of the large ORF of Jasper-IPNV has 972 amino acids, with a predicted Mr of 106465 (Duncan & Dobos, 1986). The initiator codon of this O R F in STC-IBDV lies in a sequence context that occurs at the initiation site in 18~o of eukaryotic m R N A s (Kozak, 1983). Preceding this initiator codon there is a stop codon (Fig. 1), which indeed demonstrates that this A T G is the start for protein synthesis. The polyprotein ORF leaves 5' and 3' non-coding regions of 96 bp and 130 bp (including the stop codon), respectively, on the STC-IBDV genome segment A (Fig. 1). Genome segment A of 002-73-IBDV had 28 and 65 nucleotides in the 5' and 3' non-coding regions, respectively (Hudson et al., 1986). Therefore, the nucleotide sequence of STC-IBDV (Fig. 1) is extended at the ends of genome segment A of IBDV by 68 nucleotides at the 5' end and 65 nucleotides at the 3' end of the plus strand. The additional sequences at the 5' end of the plus strand of genome segment A of STC-IBDV contain a smaller O R F of at least 435 bp that predicts a 145 amino acid protein of M~ 16550 (Fig. 1). Genome segment A of Jasper-IPNV contains a similar O R F (444 bp), which predicts a 148 amino acid protein of Mr 17335. The initiator codon for this ORF in the STC-IBDV genome segment A is probably at nucleotide position 63 in the 5' non-coding region of the polyprotein ORF (Fig. 1). This initiator codon is not preceded by a terminator codon, but is located at a position comparable to the one found in genome segment A of Jasper-IPNV (nucleotide position 68) and neither conforms to Kozak's consensus sequence (Kozak, 1983). This methionine codon begins six nucleotides upstream from the 5' end of the published 002-73-IBDV c D N A sequence (Hudson et al., 1986), indicating that genome segment A of 002-73-IBDV may also contain the small ORF. Translation in reading

q~*k

"t G*C

:?

T~

.¢ *T

C*G

.A*T A*T

.C*.U

c

T,

g,

9 G

~

G

T

"c-- ~*~ A*T T,~A

'i'*A

.e'

,c,e, c c, ~ T T A ~ d 'A

K

"C-K Fig. 2. The cDNA nucleotide sequence and the potential secondary structure (Gouy, 1987)of the 3' terminus of the plus strand of genome segment A of STC-IBDV. The direction of translation (5' to 3') of the polyproteinis indicated by the arrow and the locationof the translation termination codon by a solid line.

S T C - I B D V genome segment A cDNA sequence

1000

1

5,I

2000

573

3262 bp

I

genome segment A sequence

16.6K

] VP2

348

A

I

B

VP4

i!

1617 ...~

656

119

I

,'•i

1740

l

D

VP3

2335 ~_...... 2541

]

C

~-.............................. ]

2997 ]

I

Small ORF

I

Polyprotein ORF

Jasper-IPNV genome segment A areas of sequence homology with

IBDV

Fig. 3. Alignment of genome segment A sequences of STC-IBDV with Jasper-IPNV using the local alignment algorithm LFASTA (Pearson & Lipman, 1988). The relationship of the four areas (A to D) of sequence homology between STC-IBDV (top line) and JasperIPNV and the bases they contain are shown in the bottom of the diagram. Solid lines indicate areas of both nucleotide and amino acid sequence homology, dashed lines indicate areas of amino acid sequence homology with no nucleotide sequence homology. Also shown in the centre of the diagram are the locations of the STC-IBDV small ORF and polyprotein ORF (with the proposed cleavage sites for VP2, VP3 and VP4; see Fig. 1). Homology at the nucleotide and amino acid levels for areas A to D is given in Table 1.

T a b l e 1. Comparison of genome segment A sequences among three birnaviruses Homology between birnavirus pairs (~)* STC-IBDV and 002-73-IBDV~ Nucleotide sequence homology Whole genome segment Polyprotein ORF 5" Non-coding sequences 3' Non-coding sequences Smaller ORF Amino acid sequence homology Polyprotein ORF Small ORF

92.6 (3129) 92.5 (3036) 100 (28) 93.8 (65) 94.9 (429) 97.4 (1012) 88.1 (143)

STC-IBDV and Jasper-IPNV

002-73-IBDV and Jasper-IPNV

55"7 54"8A 59'5 B 55'1 c

55'1 54"1A 60"4~ 55"8c

(1397) (1206)~ (543) (468)

(1390) (1391) (502) (466)

0 0

0 0

60'3 B (238)

61"6B (218)

41'7A'8(499) 32"1c (224) 20"8° (279)§ 28'9 B (114)

40"9A'B(499) 33"0c (224) 21'8 D (280)§ 30"7B (114)

* Values indicate percentage of homology; figures in parentheses indicate length of nucleotide (bp) overlap or amino acid overlap. t Homology values between STC-IBDV and 002-73-IBDV are for the whole genome segment A of 002-73IBDV overlap. :~ A, B, C and D represent areas shown in Fig. 3. § Indicates amino acid sequence homology without any corresponding nucleotide sequence homology.

f r a m e 2 o f t h e plus s t r a n d o f 0 0 2 - 7 3 - I B D V g e n o m e s e g m e n t A r e v e a l s a s m a l l O R F o f 429 b p at t h e 5' e n d , w h i c h p r e d i c t s a 143 a m i n o a c i d p r o t e i n o f M r 16425. I f t h e plus s t r a n d o f g e n o m e s e g m e n t A o f 0 0 2 - 7 3 - I B D V c o n t a i n s a d d i t i o n a l n u c l e o t i d e s e q u e n c e s at its 5' e n d this

O R F m a y be a n a l o g o u s to t h e s m a l l O R F o f g e n o m e segment A of STC-IBDV. T h e a d d i t i o n a l s e q u e n c e s at t h e 3' e n d o f t h e plus s t r a n d o f g e n o m e s e g m e n t A o f S T C - I B D V c o n t a i n a 66 b p s e q u e n c e s p a n n i n g n u c l e o t i d e p o s i t i o n s 3 t 97 to 3262,

574

F. S. B. Kibenge, D. J. Jackwood and C. C. Mercado

which shows inverted complementarity relative to nucleotide positions 3166 to 3190 (Fig. 1). This inverted repeat sequence can be arranged in a hairpin configuration, as depicted in Fig. 2.

Comparison of STC-IBD V, 002-73-IBD V and JasperI P N V genome segment A nucleotide sequences and translation products

The overall nucleotide sequence homology between STC-IBDV and 002-73-IBDV genome segment A is 92-6~ in a 3129 nucleotide overlap (92-5~ in the polyprotein coding sequences) (Table 1). When genome segment A sequences of IBDV and IPNV are compared using the same library search program (FASTA) only a partial overlap (about 55~o nucleotide sequence homology) limited to 5' sequences is obtained. However, using the local alignment algorithm program (LFASTA) (Pearson & Lipman, 1988) reveals additional areas of homology between IBDV and IPNV genome segment A sequences, as shown in Table 1 and Fig. 3. There is no apparent nucleotide sequence homology between IBDV and IPNV in the VP4 region (Fig. 3). The genome segment A sequences of the two IBDV strains also contain several regions of unmatched nucleotides/indels (Collins & Coulson, 1987), relative to the Jasper-IPNV genome segment A, within the overlapping sequences. The stop codon of the STC-IBDV polyprotein ORF is TGA (Fig. 1), whereas those for the 002-73-IBDV and Jasper-IPNV polyprotein ORFs are both TAA (Duncan & Dobos, 1986; Hudson et al., 1986). The two IBDV polyproteins are more closely related than the nucleotide sequence homology would indicate, exhibiting only 2-6 ~o amino acid mismatches (97.4~o amino acid sequence homology) (Table 1). Most of the nucleotide mismatches (83.2~o) are in third base codon positions, the majority of which do not cause amino acid changes. Comparison of the amino acid sequences between either STC-IBDV or 002-73-IBDV and Jasper-IPNV polyproteins shows homology in residues 8 to 498 at the N terminus (areas A and B in Fig. 3; 41-7~o amino acid sequence homology between STC-IBDV and JasperIPNV and 40-9~o between 002-73-IBDV and JasperIPNV) and in residues 776 to 997 at the C terminus (area C in Fig. 3; 32.1~o amino acid sequence homology between STC-IBDV and Jasper-IPNV and 33.0~ between 002-73-IBDV and Jasper-IPNV) of the IBDV polyprotein (Table 1). The 8 to 498 residue segment of the IBDV polyprotein is in VP2, whereas the 776 to 997 residue region is in VP3. Within VP2 most of the amino acid sequence homology between IBDV (both STC and

002-73) and IPNV is confined to the N-terminal third and C-terminal third of the molecule (Fig. 1). There is an area of sequence analogy in the polyproteins of IBDV and IPNV detectable at the amino acid level but not at the nucleotide level due to the redundancy of the genetic code. This area (Fig. 3, area D) corresponds, in part, to VP4 sequences (20"8~o amino acid sequence homology between STC-IBDV and Jasper-IPNV and 21-8~ between 002-73-IBDV and JasperIPNV) spanning residues 556 to 829 of the IBDV polyprotein (Table 1). The amino acid sequence homology between the two IBDV strains for the small ORF protein is lower (88.1 ~) than their nucleotide sequence homology (94-9~). The majority of nucleotide mismatches in the small ORF protein (77"3~o) occur in the first base codon positions that cause over 85 ~ of the amino acid changes. All the amino acid mismatches are confined to the central region of the proteins. The amino acid sequence homology between the IBDV and IPNV small ORF proteins is about 30~ (Table 1) and limited to the central and C-terminal regions of the proteins; there is no homology in the sequences spanning residues 1 to 31 of the STC-IBDV protein. Overall the three small ORF proteins also differ in other characteristics; the STC-IBDV protein is acidic, has a net charge of - 2 at neutral pH and is rich in arginine, serine and proline, the 002-73-IBDV has no net charge at neutral pH, but is also rich in arginine, serine and proline, whereas the Jasper-IPNV protein is basic, has a net charge of + 9 and is rich only in arginine.

Discussion In this report we present the primary nucleotide sequence and deduced translation of genome segment A of the highly pathogenic American STC-IBDV. Sequence comparisons of genome segment A of STC-IBDV were made with published sequences of the homologous genome segment of 002-73-IBDV (Hudson et al., 1986) and Jasper-IPNV (Duncan & Dobos, 1986). We identified additional sequences at both the 5' and 3' ends of the plus strand in genome segment A of STC-IBDV. In the additional 5' sequences we found a second ORF predicting a 16.6K protein comparable to the one reported for Jasper-IPNV (Duncan et al., 1987) and suggest that this ORF may be common to all birnaviruses. In the 3' sequences we identified a Y-terminal inverted and complementary repeat sequence, which

STC-IBDV

predicts a hairpin structure stabilized by at least 47 bp (Fig. 2). However, since this sequence was not confirmed by direct genomic RNA analysis we cannot discount the possibility that the hairpin structure may indicate a cloning artefact, the T-terminal sequence having folded back and copied itself. If this structure does exist in vivo its location would suggest a novel expression strategy for IBDV. The 3262 bp determined for STC-IBDV genome segment A is close to the estimated total length of 3300 bp for this genome segment (Muller & Nitschke, 1987a), but may not be the real length, since both ends were not completed by direct RNA sequencing. Our ability to obtain a more complete cDNA library of the IBDV genome may have been due to the method we used to synthesize cDNA in cloning the STC-IBDV genome, which utilized RNase H (Gubler & Hoffman, 1983) and avoided the use of S1 nuclease (Jackwood et aL, 1989). The digestion with S1 nuclease is difficult to control and can be a major cause of low cloning efficiencies and the loss of a significant amount of sequence information (Gubler & Hoffman, 1983; Kimmel & Berger, 1987). The comparison of genome segment A sequences showed STC-IBDV to be very closely related to 002-73IBDV, particularly at the amino acid sequence level of the precursor polyproteins, which were 97.4~ homologous with only 26 mismatches (2.6~). Moreover, only three (11"5~o) of these mismatches were non-conservative substitutions and were all located in the VP2 molecule (Fig. 1). Over half of the amino acid mismatches between the two IBDV polyproteins are in VP2, mostly (11 out of 14) in the area of the conformationdependent epitope (residues 206 to 350 of the 002-73IBDV polyprotein) recognized by the virus-neutralizing monoclonal antibody 17/82 (Azad et al., 1987). More extensive differences were revealed when genome segment A sequences of the two IBDV strains were compared to Jasper-IPNV. IBDV and IPNV precursor polyproteins were most closely related within VP2 sequences (about 40~ amino acid sequence homology), followed by VP3 sequences (about 32~o amino acid sequence homology). Within the VP2 molecule the conformation-dependent epitope region was again the least homologous. The amino acid heterogeneity in this region between IBDV and IPNV was more conspicuous than between the two IBDV strains, which is not surprising since IBDV and IPNV are antigenically distinct birnaviruses (Dobos et al., 1979). Previously, Azad et al. (1987) noted extensive homology, at both the nucleotide and the amino acid level, between the N-terminal and C-terminal polypeptides of the 002-73-IBDV and Jasper-IPNV, but very little homology between the internal VP4s. Our comparisons

genome segment A c D N A sequence

575

using the local alignment aligorithm LFASTA (Pearson & Lipman, 1988) also show that IBDV and IPNV polyproteins are least related within VP4 sequences, being only about 21~ homologous at the amino acid level, with no homology at the nucleotide level. This high sequence divergence within VP4 of two viruses belonging to the same genus is unexpected, since VP4 is reported to be involved in precursor polyprotein processing (Azad et al., 1987; Duncan et al., 1987; Jagadish et al., 1988). The complete lack of nucleotide sequence homology within the non-coding regions of IBDV and IPNV is surprising and difficult to explain because both viruses belong to the same genus and their genomes show perfect homology in organization and function. In fact, one would expect fewer similarities among coding sequences, but significant similarities in the non-coding portions of the viral genomes, as occurs in other RNA viruses, because of the common involvement of the non-coding regions in the virus-specific processes of transcription, translation, replication and assembly. For instance, Bunyaviridae viruses have conserved sequences at the 3' termini of the L, M and S virion RNA species of viruses belonging to the same genus, but different sequences for viruses representing other genera (Clerx-Van Haaster et al., 1982). A similar situation occurs in the Reoviridae family, where the conserved terminal sequences are identical for all members of a genus but differ from genus to genus (Antczak et al., 1982; Li et al., 1980; Omura et al., 1988). Picornaviruses also show significant homology for viruses belonging to a particular genus, whereas viruses representing different Picornaviridae genera have little T-terminal sequence similarity (Porter et al., 1978). One possible explanation in the case of the birnaviruses examined in this study is that the terminal nucleotide sequences, which may be conserved in members of the birnavirus genus, are missing in the published sequences of genome segment A, since both ends of this segment have not been completed by direct RNA sequencing (Hudson et al., 1986; Duncan & Dobos, 1986). Our findings also indicate a need for the possible division of the family Birnaviridae into more than one genus. In summary, we have determined 3262 bp of genome segment A of STC-IBDV and identified a second ORF in the 5' sequences and a potential hairpin structure in the 3' sequences of the plus strand of genome segment A of STC-IBDV. The comparisons described in this paper indicate the conserved regions of the genome segment A proteins that should be logical targets for specific mutagenesis in order to delineate functional domains within these proteins. More sequence information, particularly of other birnaviruses such as drosophila X

576

F. S. B. Kibenge, D. J. Jackwood and C. C. Mercado

virus of the fruit fly, is needed to clarify further the evolutionary relationships between birnaviruses and to see whether nucleotide sequence data justify the grouping of these viruses into one genus.

The authors thank Drs David A. Benfield, Kenny V. Brock, Erwin M. Kohler and Kenneth W. Theil for critical reading of the manuscript. This work was supported by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript number 185-89 of the Ohio Agricultural Research and Development Center. The sequence data reported will appear in the DDBJ, EMBL and GenBank databases under the accession number d00499.

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FAHEY, K. J., ERNY, K. & CROOKS, J. (1989). A conformational immunogen on VP-2 of infectious bursal disease virus that induces virus-neutralizing antibodies that passively protect chickens. Journal of General Virology 70, 1473-1481. GouY, M. (1987). Secondary structure prediction of RNA. In Nucleic Acid and Protein Sequence Analysis: A Practical Approach, pp. 259284. Edited by M. J. Bishop & C. J. Rawlings. Oxford & Washington, D.C. : IRL Press. GRUNSTEIN, M. & HOGNESS, D. S. (1975). Colony hybridization: a method for isolation of cloned DNAs that contain a specific gene. Proceedings of the National Academy of Sciences, U.S.A. 75, 39613965. GUBLER, U. & HOFFmAN, B. J. (1983). A simple and very efficient method for generating cDNA libraries. Gene 25, 263-269. HOLMES, D. S. & QUIGLEY, M. (1981). A rapid boiling method for the preparation of bacterial plasmids. Analytical Biochemistry 114, 193197. 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. ISMAIL, N. M., SAIF, Y. M. & MOORHEAD, P. D. (1988). Lack of pathogenicity of five serotype 2 infectious bursal disease viruses in chickens. Avian Diseases 32, 757-759. JACKWOOD, D. J., KIBENGE,F. S. B. & MERCAOO, C. C. (1989). The use of biotin labeled cDNA probes for the detection of infectious bursal disease viruses. Avian Diseases (in press). JAGAOtSH, M. N., STATON,V. J., HUDSON, P. J. & AZAD, A. A. (1988). Birnavirus precursor polyprotein is processed in Escherichia coli by its own virus-encoded polypeptide. Journal of Virology 62, 10841087. KIBENGE, F. S. B., DrnLLON, A. S. & RUSSELL, R. G. (1988). Biochemistry and immunology of infectious bursal disease virus. Journal of General Virology 69, 1757-1775. KXMmEL, A. R. & BERGER, S. L. (1987). Preparation of cDNA and the generation of cDNA libraries: overview. Methods in Enzymology 152, 307-316. KOZAK, M. (1983). Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. MicrobiologicalReviews 47, 1-45. LI, J. K., KEENE, J. D., SCHEIBLE,P. P. & JOKLIK, W. K. (1980). Nature of the 3' terminal sequences of the plus and minus strands of the S1 gene of reovirus serotypes 1, 2 and 3. Virology 105, 41-51. MACDONALD, R. D. & DOBOS, P. (1981). Identification of the proteins encoded by each genome segment of infectious pancreatic necrosis virus. Virology 114, 414-422. MANIATIS, T., FRITSCH, E. F. & SAMBROOK, J. (1982). Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory. MORGAN, M. M., MACREADIE,I. G., HARLEY, V. R., HUDSON, P. J. & AZAD, A. A. (1988). Sequence of the small double-stranded RNA genomic segment of infectious bursal disease virus and its deduced 90-kDa product. Virology 163, 240-242. MULLER, H. & NITSCHKE, R. (1987a). Molecular weight determination of two segments of double-stranded RNA of infectious bursal disease virus, a member of the birnavirus group. Medical Microbiology and Immunology 176, 113-121. MULLER, H. & NITSC/-mE, R. (1987b). The two segments of infectious bursal disease virus are circularized by a 90,000 Da protein. Virology 159, 174-177. OMURA, T., MINOBE,Y. & TSUCHIZAKI,T. (1988). Nucleotide sequence of segment $10 of the rice dwarf virus genome. Journal of General Virology 69, 227-231. PEARSON, W. R. & LIPMAN, D. J. (1988). Improved tools for biological sequence comparison. Proceedings of the National Academy of Sciences, U.S.A. 85, 2444-2448. PORTER, A. G., FELLNER, P., BLACK, D. N., ROWLANDS,D. J., HARRIS, T. J. R. & BROWN, F. (1978). Y-Terminal nucleotide sequences in the genome RNA of picornaviruses. Nature, London 276, 298-301. RAYMOND, G. J., BRYANT, P. K., III, NELSON, A. & JOrlNSON, J. D. (1988). Large-scale isolation of covalently closed circular DNA using gel filtration chromatography. Analytical Biochemistry 173, 125-133.

S T C - I B D V genome segment A c D N A sequence

SPIES, U., MULLER, H. & BECHT, H. (1987). Properties of RNA polymerase activity associated with infectious bursal disease virus and characterization of its reaction products. Virus Research $, 127140. WINTERFIELD, R. W. & THACKER, H. L. (1978). Immune response and

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pathogenicity of different strains of infectious bursal disease virus applied as vaccines. Avian Diseases 22, 721-731.

(Received 31 July 1989; Accepted 23 October 1989)