The Complete Nucleotide Sequence of the Egg Drop Syndrome Virus: an Intermediate Between Mastadenoviruses and Aviadenoviruses

VIROLOGY 238, 145–156 (1997) ARTICLE NO. VY978815

The Complete Nucleotide Sequence of the Egg Drop Syndrome Virus: An Intermediate between Mastadenoviruses and Aviadenoviruses

Michael Hess,*,1 Helmut Blo¨cker,† and Petra Brandt†

*Institut fu¨r Geflu¨gelkrankheiten, Freie Universita¨t Berlin, Koserstrasse 21, 14195 Berlin, Germany; and †Gesellschaft fu¨r Biotechnologische Forschung mbH, Mascheroder Weg 1, 38124 Braunschweig, Germany Received May 7, 1997; returned to author for revision June 11, 1997; accepted September 2, 1997

The complete nucleotide sequence of an avian adenovirus, the egg drop syndrome (EDS) virus, was determined. The total genome length is 33,213 nucleotides, resulting in a molecular weight of 21.9 1 106. The GC content is only 42.5%. Between map units 3.5 and 76.9, the distribution of open reading frames with homology to known genes is similar to that reported for other mammalian and avian adenoviruses. However, no homologies to adenovirus genes such as E1A, pIX, pV, and E3 could be found. Outside this region, several open reading frames were identified without any obvious homology to known adenovirus proteins. In the region organized similarily as other adenoviral genomes, most homologies were found to an ovine adenovirus (OAV strain 287). The highest level of amino acid identity was found for the hexon proteins of EDS and OAV. The virus-associated RNA (VA RNA) was identified thanks to the homology with the VA RNA of fowl adenovirus serotype 1 (FAV1). Similarities with FAV1 were also found in the fiber protein. Our results demonstrate that the avian EDS virus represents an intermediate between mammalian and avian adenoviruses. The nucleotide sequence and genomic organization of the EDS virus reflect the heterogeneity of the aviadenovirus genus and the Adenoviridae family. ᭧ 1997 Academic Press

INTRODUCTION base like all mastadenoviruses and like group II avian adenoviruses (Valentine and Pereira, 1965; Van den The family of Adenoviridae is composed of two differ- Hurk, 1992). Thus it differs from group I of avian adenovi- ent genera: the aviadenoviruses infecting birds and the ruses which have two fibers per penton base (Gelder- mastadenoviruses infecting mammals. Infections by blom and Maichle-Lauppe, 1982). At 42 nm, the fiber has some avian adenoviruses are directly correlated with the nearly the same length as the long fiber of FAV1. EDS occurrence of a specific disease (Monreal, 1992). These does hemagglutinate chicken erythrocytes, which is a are the egg drop syndrome 76 in hens (group III), the useful diagnostic method for separating EDS from group hemorrhagic enteritis of turkeys, and the marble spleen I avian adenoviruses. disease of pheasants (group II). The conventional adenoviruses (group I) are isolated from chickens (FAV1-12), The EDS genome length of 35 kb which was deter- geese (GAV1-3), ducks (DAV2), and turkeys (TAV1-2). One mined by restriction enzyme analysis is in the range of serotype is isolated from pigeons (Hess et al., 1997). A mastadenovirus genomes (Kisary and Zsak, 1980). Inves- possible pathogenic significance of the conventional tigation of the genomic relationship between EDS strains avian adenoviruses remains quite limited, but some FAV and FAV1 revealed variable DNA restriction patterns strains are involved in the occurrence of an inclusion (Todd et al., 1988; Zsak and Kisary, 1981). Using Southern body hepatitis in broilers (Erny et al., 1991). blot technique, Zakharchuk et al. (1993) found no homol- The egg drop syndrome (EDS) virus is the sole member ogy of EDS with FAV1 and human adenovirus (HAd) sero- of group III avian adenoviruses. It is serologically not type 5, but homologous sequences were found with sev- related to group I and group II avian adenoviruses eral bovine adenovirus (BAV) serotypes. (McFerran, 1981a). Only one serotype is known, although Besides vertical transmission, a horizontal spread was restriction enzyme analysis shows minor variations observed with EDS (Cook and Darbyshire, 1980). In some among different isolates (Todd and McNulty, 1978). cases, the horizontal transmission occurred from water- The EDS virus was classified as an adenovirus in view fowl to chickens. The presence of virus and of antibodies of its morphology, replication, and chemical composition. in ducks might indicate their role as primary host where- The virion is an icosahedron with a size of 80 nm (Kraft fore the EDS virus is classified as duck adenovirus type et al., 1979). The EDS virus has only one fiber per penton 1 (DAV1) (Russell et al., 1995). Antibodies were also found in different other avian species but a specific dis- 1 To whom correspondence and reprint requests should be ad- ease is reported only for chickens, indicating that EDS dressed. Fax: 49 30 8385824. E-mail: [email protected]. crosses the species barrier (for a review see McFerran,

0042-6822/97 $25.00 145 Copyright ᭧ 1997 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. Comparison of the genome organization of HAd2, EDS, and FAV1. Open reading frames of the upper, rightward transcribed strand are indicated on top and for the complementary, leftward transcribed strand at the bottom. Filled boxes indicate unassigned ORFs ú100 aa. Abbreviations: EP, endoproteinase; DBP, DNA binding protein; pTP, precursor terminal protein; POL, DNA polymerase. Data for HAd2 and FAV1 were taken from accession numbers J01917 and U46933.

1991). Although chickens of each age can be infected, performed according to Rohn et al. (1997). Purified viral the clinical signs are restricted to the laying period, which DNA was partially digested with Tsp5091 and Sau3A. is probably due to the reactivation of latent virus (McFer- Restriction fragments of 1–3 kb were extracted from an ran et al., 1978). Major clinical symptoms are thin shelled 0.5% agarose gel with the gel extraction kit (Qiagen, eggs, loss of color in pigmented eggs, and a temporarily Qiaex). Isolated fragments were cloned into EcoRI- or reduced egg production (Van Eck et al., 1976). BamHI-digested pUC19 vector, treated with alkaline Some nucleotide sequences are available from group phosphatase, according to standard protocols (Sam- I avian adenoviruses. The complete nucleotide sequence brook et al., 1989). The nucleotide sequence of cloned of the fowl adenovirus serotype 1 (FAV1 or CELO) was viral fragments was determined by using fluorescence- published recently (Chiocca et al., 1996). In addition, sev- labeled primers in combination with the cycle sequenc- eral genes of FAV10 were also sequenced. Jucker et al. ing method. Viral DNA was used to complete the terminal (1996) published the sequence of the penton base gene ends of the sequence. The reported sequence is the and core proteins of the hemorrhagic enteritis virus (HEV) result of at least three sequencing reactions. For se- which belongs to group II avian adenoviruses. Some sequence analysis, the GCG, PC Gene, and Genmon soft- quences of the EDS virus which include inverted terminal ware packages were used. Homology studies were done repeats (Shinagawa et al., 1987), penton base, pVIII with FASTA and BLAST programs; sequence alignments (Rohn et al., 1997), and protease gene (Harrach et al., were done with CLUSTAL V and PALIGN. Secondary 1997) have been published. structures of RNAs were predicted by MFOLD. The final In the present report, we have completed the whole sequence was submitted to the EMBL Databank (acces- sequence of the EDS virus. The major aim was to deter- sion number Y09598). mine the relationship of EDS to other adenoviruses. Fur- The following data bank entries were taken for homol- thermore, the sequence data might be helpful in investi- ogy studies: HAd serotype 2, J01917; HAd12, X73487; gation of the in vivo behavior and epidemiology of the HAd40, L19443; FAV serotype 1, U46933 and X84724; virus. FAV10, U26220, P32538, L084500, U26221, and P36856; HEV, U31805; ovine adenovirus (OAV) serotype 5, MATERIAL AND METHODS U40839, U31557, U40837, and U18755; BAV serotype 3, The propagation and purification of the EDS virus P03278, P19119, U08884, and Q03553; canine adenovi- strain127, followed by extraction of the viral DNA was rus (CAV) serotype 1, U55001; mouse adenovirus (MAV)

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TABLE 1 Feature Table of EDS Genes and Encoded Proteins

EP cleavage Start a Stop aab MW sites Poly(A) signal

31.8-kDa proteinc 1,111 251 286 31,786 6, 209, 225 121 E1B E1BS 1,175 1,651 158 18,049 E1BL 1,676 2,857 393 42,756 2,909 E2B IVA2 4,112 2,907 401 45,758 2,898 POL 7,219 3,980 1079 123,868 pTP 8,943 7,198 581 67,106 156, 265 L1 52K 9,035 10,051 338 38,273 11,639 IIIa 10,017 11,744 575 63,880 548 L2 III 11,774 13,132 452 51,105 pVII 13,182 13,664 160 18,011 mu 13,683 13,886 67 7,344 37 14,539 L3 pVI 13,942 14,634 230 24,315 28,219 Hexon 14,656 17,388 910 102,215 EP 17,385 17,993 202 22,935 18,219 E2A DBP 19,168 18,005 387 43,122 18,005 L4 100K 19,217 21,346 709 80,002 pVIII 21,687 22,439 250 28,501 110, 148, 178 22,484 L5 Fiber 22,685 24,619 644 67,678 24,638 E4 34-kDa protein 25,565 24,678 295 34,376 24,682 VA RNA 30,376 30,475

a Start is the first base in ATG, except VA RNA. b Amino acids. c Homology with OAV287 p28K protein. serotype 1 P12535, P12536, P12539, X74742, P19721, al., 1996). The GC content of the inverted terminal re- P19722, and P48308. peats, the viral protease, penton base, DNA binding protein, hexon, 100K, and the IVA2 genes are below average. RESULTS AND DISCUSSION After translation of the nucleotide sequence into the appropriate open reading frames (ORFs), typical adenovi- General features of the EDS genome rus proteins were identified in the genomic region ex- The overall genome length is 33,213 bp. This value is tending from 1 to 25 kb (Fig. 1, Table 1). It includes two in good agreement with the molecular weight of 22.9 1 ORFs with very weak homology to the E1B proteins of 106 Da determined for the EDS strain B8/78 by Kisary other adenoviruses, a genomic region not identified in and Zsak (1980) using restriction enzyme analysis. Thus FAV1. Only one unassigned ORF was identified at the the genome is more than 10 kb smaller than the length left end of the genome, while several unidentified ORFs of 43.8 kb which was reported for the avian FAV1 genome can be found in the FAV1 genome. At the right end, an (Chiocca et al., 1996). E4 34-kDa protein adjacent to the fiber gene was found, Human adenoviruses have been classified in five dif- localized similarly as in HAd2. Downstream of the E4 ferent subgroups according to the GC content of their protein several ORFs were found without obvious homol- DNA ranging from 48 to 59% (Pina and Green, 1965). This ogy with any adenovirus proteins in the database. correlates with the serological classification system. In FAV1, the homologous region to HAd2 extends from The EDS DNA has a rather low GC content of 42.5%, the IVA2 gene and ends rightward at the pVIII protein which is nearly 12% lower than that of FAV1 and 7% (Fig. 1). The location of pVIII in HAd2 and FAV1 genomes below the level of HAd12. A much lower GC content of is nearly identical, whereas the pVIII protein in EDS is 33.6% is reported only for the ovine adenovirus (Vrati et shifted 5 kb leftward. This shift is due to two facts. First,

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FIG. 2. Nucleotide sequence comparison of the EDS and OAV287 genomes using the GENMON graphic program (blocks of 9 identical nucleotides are included). in FAV1 and HAd2 genomes, several proteins which do Inverted terminal repeats not occur in EDS are located upstream of the IVa2 gene. Second, 100K and DBP genes which are located in the With 53 nucleotides (nt) the ITRs are very short (Fig. middle of EDS genome are smaller than their counter- 3). Two different bases and one additional base at the parts in HAd2 and FAV1 (Fig. 1). 5؅ end of the ITRs were detected in comparison to the The nucleotide sequence homology was determined already reported sequence (Shinagawa et al., 1987). As for EDS and all known adenovirus genomes. The highest in most adenoviruses, the ITRs of EDS starts with dC homology was seen for EDS and ovine adenovirus 287 (the ITRs of FAV1 start with dG (Alestro¨m et al., 1982)). (OAV287) (Vrati et al., 1996) (Fig. 2). Nucleotide sequence As for other adenoviruses, the ITRs of avian adenovi- homology starts nearly at the beginning and ends at 22 ruses can be divided into an AT-rich region followed by kb. The homology between EDS and FAV1 (Chiocca et a GC-rich part. The AT-rich region in the EDS ITRs in- al., 1996), HAd2 (Roberts et al., 1984), HAd12 (Sprengel cludes the first 39 nt and contain 66.6% AT. In HAd2, this et al., 1994), and HAd40 (Davison et al., 1993) is much region contains sequences which are necessary for the lower. initiation of DNA replication (Wides et al., 1987). The 9ATT-

FIG. 3. Nucleotide sequence of the inverted terminal repeats of the EDS and FAV1 DNA. Identical nucleotides are indicated by É. GC-rich regions are shown in italics and highly conserved sequences in AT-rich parts are in bold.

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FIG. 4. Secondary structure models for VA RNAs. The structures for EDS and FAV1 VA RNAs were derived by computer analysis using the MFOLD program of the GCG package. Identical nucleotides of both VA RNAs are shown in bold. The conserved tetranucleotides GGGU and ACCC are boxed.

AATAA16 sequence in EDS ITRs could probably be an the VA RNAs of FAV1 and EDS. The EDS VA RNA resem- equivalent sequence motif (Fig. 3). Adenovirus DNA repli- bles typical features of VA RNAs, i.e., a 5؅-terminal G and -cates via a jumping-back mechanism (King and van der four T at the 3؅ end (Fig. 4). In common with all mamma Vliet, 1994), resulting in a repetition of 2–4 nucleotides lian adenoviruses, the avian VA RNAs contain the com- at the terminal ends. Such a terminal repetition is missing plementary tetranucleotides GGGU and ACCC in the cen- in EDS, PAV4 (Reddy et al., 1995), OAV287 (Vrati et al., tral domain. The VA RNA is implicated with an efficient 1996), and HAd19 (Shinagawa et al., 1987) indicating a protein synthesis during viral growth by interacting with modification of the proposed replication model for these the cellular p68 kinase (Katze et al., 1987). This function viruses. The GC-rich region (57% GC) containing 14 nu- was found to be mediated by the central domain of the cleotides is very small and the GGGNGG sequence rec- VA RNA together with the apical stem (Ghadge et al., ognized by the transcription factor SP1 in mammalian 1994). These parts are indeed the most conserved ones adenovirus ITRs is not present in the ITRs of avian adeno- between the EDS and FAV1 VA RNA (Fig. 4). viruses. Ma and Mathews (1996) postulated that the adenovirus VA RNAs probably originate from cellular sequences. Virus associated (VA) RNA Since FAV1 and EDS can multiply in common hosts, the similarity of the VA RNAs of EDS and FAV1 might be an All so far identified VA RNAs of mammalian adenovi- argument in support of this hypothesis for avian adenoviruses which are between 150 and 170 nucleotides long ruses. However, it seems that an insertion of such cellu- are transcribed rightward and are localized at map posi- lar sequences in the EDS and FAV1 genomes occurred tion (mp) 30 (Ma and Mathews, 1993). Contrary to this, in different directions. the VA RNA in FAV1 is shorter, located at mp 90 and is transcribed leftward (Larsson et al., 1986). Although the Early regions of the genome VA RNA in EDS is located similar to FAV1 at mp 90, it is transcribed rightward as in mammalian adenoviruses E1 region. No E1A region could be identified. A unique (Fig. 1). With 100 and 91 nt, respectively, the EDS and small ORF of 69 aa could be found in the first 1.2 kb of FAV1 VA RNAs are very short. The overall nucleotide the upper strand with no homologies to other adenovirus sequence homology of EDS with FAV1 DNA is very low. proteins. An 31.8-kDa ORF was identified on the comple- In contrast to this, a 74% identity was detected between mentary strand transcribed leftward with 31% identity to

AID VY 8815 / 6a53$$$182 10-21-97 04:14:10 vira AP: VY 150 HESS, BLO¨ CKER, AND BRANDT a protein which is located roughly at the same position in the OAV287 genome. For OAV287, this protein was shown to be a precursor of a structural protein (Vrati et al., 1996). Several adenovirus proteins are cleaved by a viral protease and two consensus motifs are proposed as cleavage sites (Webster et al., 1989). The 31.8-kDa protein of EDS has three consensus cleavage sites at positions 6, 209, and 225, which suggests that this might be a putative structural protein as in OAV287. The first rightwardly transcribed ORF of the upper strand with more than 100 aa starts at position 1175 and stops at 1651 (Table 1). Together with the following protein of 393 aa, these two ORFs represent probably the E1B region (Figs. 5a and 5b). A possible E1B promoter is located at position 996–1010 and the transcripts terminate at the AATAAA polyadenylation signal at 2909–2914. The small (17.5 kDa) E1B protein shows only weak homology to E1B proteins of other adenoviruses (Table 2). Pronounced similarities to E1B genes of OAV287 were found in the large E1B protein (Fig. 5b). In common with FAV1 and OAV287, the EDS genome has no pIX gene, which in human adenoviruses follows the E1B region. E2 region. The E2 region comprises a set of proteins involved in adenovirus DNA replication. These are the DNA-binding protein (DBP), the preterminal protein (pTP), and the DNA polymerase (POL), transported into the nu- cleus via nuclear localization signals (NLS) (De Robertis et al., 1978; Zhao and Padmanabhan, 1988). The EDS DBP is localized between nt 18005 and 19168. The resulting protein consisting of 378 aa is 151 aa shorter than the DBP of HAd2 and this truncation can be found mainly in the N-terminus (Fig. 6a). The size of the DBP and the deletion sites are similar to the DBP of OAV287. Interestingly, at the C-terminus of the EDS and OAV287 DBPs, a sequence which consists mainly of neg- atively charged glutamic acids and aspartic acids can be seen. These parts show no homology to the respective FAV1 and HAd2 DBPs (Fig. 6b). A basic sequence which is homologous to the NLS sequence of the SV40 large T antigen (Kalderon et al., 1984) is located in this C-terminal extension of the EDS and OAV287 DBP (Fig. 6b). The 380RLPVRRRRRRVP391 sequence in the HAd2 pTP was also identified as a NLS sequence motif (Ramachandra FIG. 5. Alignment of putative E1BS (a) and E1BL (b) proteins of EDS, and Padmanabhan, 1995) and a homologous sequence OAV287, and HAd2 using the Clustal V program (parameters: open gap 308 319 TLPARTRRTRRP can be found in the EDS pTP. cost 10, unit gap cost 10). Identical amino acids in all three proteins E3/E4 region. In human, canine, bovine, and mouse are indicated by an asterisk and similar amino acids are indicated by adenoviruses the E3 region is located between pVIII and dots. Identical amino acids in the EDS and OAV287 E1B proteins are the fiber protein and it codes for a set of diverse proteins given in bold. (Wold and Gooding, 1991). In EDS, the fiber gene starts only 45 nucleotides after the pVIII gene and no known E3 1). Two sets of ORFs are transcribed from the lower strand region can be identified. The lack of an E3 region between leftward and one, localized between, is transcribed pVIII and fiber gene is also reported for FAV1 and OAV287. rightward from the leading strand. The ORF localized im- (Chiocca et al., 1996; Vrati et al., 1995). In common with mediately after the fiber protein codes for a protein of 34.4 EDS, both of these viruses have different unassigned kDa. This protein has significant homology to the E4 34- ORFs at the right end of the genome. In EDS three blocks kDa protein of HAd2 and to respective E4 proteins of other of ORFs are present downstream of the fiber protein (Fig. adenoviruses (Table 2). The function of the other ORFs

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TABLE 2 Amino Acid Sequence Identity between Identified Genes of EDS Virus and Known Genes of Other Avian and Mammalian Adenoviruses

FAV1 FAV10 HAd2 HAd12 HAd40 OAV287 BAV3 CAV1 MAV1 HEV

E1BS *a — b 20c 18 22 32 —2421— E1BL * — 24 23 24 41 —2122— IVA2 36 — 42 41 41 68 —4242— POL 40 — 46 46 48 56 —49—— pTP 35 35 35 37 38 54 —36—— 52K 36 — 32 34 32 45 —32—— IIIa 27 — 34 34 34 53 —31—— III 55 53 57 56 56 69 —57—52 pVII 46 44 37 39 39 49 —30—32 mu 52 58 45 46 48 55 — 51 — 43 pVI 36 — 33 34 33 49 —2430— Hexon 52 51 54 55 56 73 53 56 52 — EP 41 — 40 41 42 57 41 41 — — DBP 30 — 35 33 34 53 —3517— 100K 39 36 36 36 34 56 —3435— pVIII 29 — 34 35 36 53 34 38 33 — Fiber 35/32d — 24 28 34/28e 29 32 25 24 — E4f * — 18 22 25 28 —2220—

aGene not identified. b Sequence not available. c % identity determined with PALIGN (PCGENE) using default parameters (open gap cost 3.0 and unit gap cost 2.0). Best homologies for each protein are in bold. d Fiber1/fiber2. e Long fiber/short fiber. f E4 34-kDa protein. is not known and the existence of a E3 region remains (1996) found that a general rearrangement of the genome speculative in the EDS genome. Remarkably, none of the resulted in an E4 region at the left end of the genome and unassigned ORFs has any homology to unassigned ORFs the VA RNA at the opposite end. This situation is totally at the right end of FAV1 or OAV287. For FAV1 Chiocca et al. different from all so far characterized mammalian adenoviruses and also different to EDS with an E4 protein at the right end close to the fiber protein. It seems that proteins downstream of the fiber genes are specific for each of these viruses regardless of virus origin. It needs to be determined if some of the unassigned EDS proteins do have any function comparable to E3. A common function of the unassigned ORFs seems to be questionable due to the lack of sequence homology.

Late regions of the genome Based on homology studies, the major late promoter is located near nt 5000 with a TATA box which centers at position 4813. A general feature of human adenoviruses is the occurrence of five different groups of late transcripts (L1–L5), each terminating with a similar polyadenylation signal (AAUAAA) (Fraser et al., 1982). This organization could be found only for the L4 transcripts of the EDS genome, whereas the polyadenylation signals of the other late transcripts show some variation. A possible polyadenylation signal for the 52k protein gene is located at map position 11,639. Protein IIIa gene termi- FIG. 6. Homologies of the N-terminus (a) and C-terminus (b) of the EDS, OAV287, FAV1, and HAd2 DBPs using the Clustal V program nates probably at position 14,539 together with the L2 (parameters and description see Fig. 5). A possible NLS sequence in penton base and core proteins genes (Table 1). The hex- the C-terminal end is underlined. anucleotide sequence AUUAAA was reported to be a

AID VY 8815 / 6a53$$$183 10-21-97 04:14:10 vira AP: VY 152 HESS, BLO¨ CKER, AND BRANDT polyadenylation signal for some E3 mRNAs of HAd2 (Ahmed et al., 1982). This sequence can be found at positions 18,219 and 24,638, terminating probably L3 and L5 transcripts, respectively. However, polyadenylation signals of EDS late transcripts must be determined ex- perimentally. Hexon. The hexon protein is the major capsid protein of adenoviruses on which type, group, and subgroup antigenic determinants are located (Norrby, 1969). An antigen shared between EDS and FAVs was reported earlier (McFerran et al., 1978) but serum neutralization, double immunodiffusion, and hemagglutination inhibition tests showed that the two groups of avian adenoviruses (I and III), to which these viruses belong, are not related (Adair et al., 1979). In these experiments the lack of the group I-specific antigen was shown by immunofluorescence assay. The sequence results confirm the serological dis- FIG. 8. Sequence alignment of the EDS and OAV287 hexon L1 and tinctness between the two avian adenovirus groups. L4 regions. The alignment was obtained with PALIGN of the PCGENE Whereas the hexon proteins of FAV1 and FAV10 have program package (see Table 2). 76% identical amino acids, the identity with the EDS hexon protein is only 52 and 51%, respectively. A remark- able 73% aa identity is between the hexon proteins of lated that species specific residues are probably located EDS and OAV287 (Table 2). in this loop (Crawford-Miksza and Schnurr, 1996). In Aligning several human adenovirus hexon proteins, agreement with this, identities of 4.2 and 8.8% only were Bailey and Mautner (1994) showed that hexons of the reported for L1 and L4 regions of different mammalian same subgenus belong to the same cluster. In the den- and FAV10 hexon proteins (Sheppard et al., 1995). Con- drogram in Fig. 7, hexon proteins of EDS and OAV287 trary to that, the EDS and OAV287 L1 and L4 regions are in the same cluster, although they originate from share 56 and 45% aa (Fig. 8). In comparison, in the same adenoviruses of two different genera. Two functional regions of EDS and FAV1, only 28 and 38% identical components of the hexon protein are described: The con- amino acids were found (data not shown). The good ho- served pedestral regions P1 and P2 and variable loops mology between the EDS and OAV287 hexon proteins in 1–4 which form the surface of the protein (Roberts et al., the loop regions support the result of the clustal align- 1986). Recently it was shown that hexon hypervariable ment shown in Fig. 7. regions (HVRs) itself showed subgenus clustering and Fiber. A protein of 644 aa starts at nucleotide 22,685 six of seven HVRs are located in the L1 loop. The greatest and is clearly the fiber protein. The MW of 68 kDa for variability between human and nonhuman serotypes the fiber protein is in good agreement with earlier find- could be found in the hexon L4 loop and it was specu- ings that a 67-kDa protein is the most abundant protein in purified hemagglutinins determined by SDS–poly- acrylamide gel electrophoresis (Todd and McNulty, 1978). Virus tropism depends to large extent on fiber protein, since this is a viral protein interacting with primary cell receptor (Philipson et al., 1968; Louis et al., 1994). Because of the important role of fiber proteins in determining cell tropism, the EDS fiber is a major factor in the spread of EDS from ducks to chickens. Like all adenovirus fiber proteins, the EDS fiber is organized in three regions, i.e., tail, shaft, and head. The tail of the EDS fiber is lacking the poly(G) stretch which occurs in the tail of the long fiber of FAV1 and FAV8 (Hess et al., 1995; Pallister et al., 1996). It was shown for HAd2 fiber that the 2KRAR5 sequence in the tail is a NLS sequence (Hong et al., 1991). In the EDS fiber the 2KRLR5 of the protein could have the same function. A 3KRLR6 FIG. 7. Dendrogram of an alignment of different adenovirus hexon sequence was also found in the fiber tails of human proteins (see Table 1) by using the CLUSTAL V program in the PCGENE subgenus D fiber proteins (Pring-Akerblom and Adrian, program package (parameters: open gap cost, 10; unit gap cost, 10). 1995). The VYPY sequence in the fiber tail is another very

AID VY 8815 / 6a53$$$183 10-21-97 04:14:10 vira AP: VY COMPLETE NUCLEOTIDE SEQUENCE OF THE EDS VIRUS 153 conserved sequence motif and was found in all mammalian adenovirus fiber proteins sequenced so far, except HAd12 (Chroboczek et al., 1995). This sequence is probably involved in the interaction with the penton base (Cail- let-Boudin, 1989; Hong and Boulanger, 1995). In common with FAV1 and FAV8 fiber genes the equivalent sequence in the EDS fiber is 11VYPF14. The fiber shaft consist of several 15 amino acid repeating motifs with conserved hydrophobic residues (Green et al., 1983). Position 8 is a proline or a glycine followed by a leucine. This organization, slightly altered, can also be found in the FAV1 fibers (Hess et al., 1995). The FAV1 fibers are made up of double and a few triple repeats. Figure 9 shows a possible organization of the EDS fiber protein. Whereas FIG. 10. Comparison of head domains of EDS fiber and FAV1 long the localization of hydrophobic residues is conserved, fiber (program: PALIGN). most of the shaft repeats consist of more than 15 amino acids. The 15 aa containing GL or PL repeat occurs in identical amino acids can be found in the two fiber heads the proposed model only three times. of the FAV1 short and long fibers. The fiber head carries The overall sequence identity with other fiber proteins the antigenic type specific determinant g which is proba- is rather low (Table 2). The long fiber of FAV1 and the bly identical to the viral hemagglutinin (Norrby, 1969). EDS fiber have 35% identical amino acids. Interestingly, EDS and FAV1 are the only avian adenoviruses with hem- the fiber head domains of both proteins have 40% identi- agglutinating activities, but there is no relation between cal amino acids (Fig. 10). Contrary to this, only a few each other, as determined by hemagglutination inhibition tests (Adair et al., 1979). In addition, EDS hemaggluti- nates erythrocytes of avian origin, whereas FAV1 hemag- glutinates only rat erythrocytes (McFerran, 1981b). The classification of the adenovirus family into two different genera, mastadenovirus and aviadenovirus (Norrby et al., 1976), has its origin in the fact that both genera do not share any immunologically cross-reacting structural protein (Kawamura et al., 1964; McFerran et al., 1972). However, alternative grouping of viruses into genera could be done according to common characteris- tics of virus species (Murphy, 1996). Taking phylogenetic relationship, genome organization, and gene order as primary parameters for classification, our results clearly show that the existing scheme remains questionable. Therefore, the aforementioned properties were taken to establish a classification model for the Adenoviridae family consisting of three different genera (Fig. 11). The HAd2 and HAd5 should be regarded as the refer- ence viruses for the classical mastadenovirus genus (genus I) due to their close sequence relationship (Chroboc- zek et al., 1992). Included in this genus are also the HAd12, HAd40, and CAV1. A common feature of genus I viruses is the presence of an E1A region, an E3 region, and pIX and pV proteins localized nearly at the same positions (Fig. 1). These regions and genes are obviously absent in the EDS virus and OAV287 representing the second genus. A further common feature of the EDS virus and OAV287 is the low GC content, being 42.5 and 33.6%, respectively. Nearly all protein homologies are 10–25% higher for EDS and OAV287 than the homology obtained FIG. 9. Organization of the EDS fiber into head shaft and tail according to Green et al. (1983) and Stouten et al. (1992). Tail regions of by comparing the same proteins with those of genera I known function which are discussed in the text are underlined. Con- and III (Table 1). EDS and OAV287 are unique, since a served hydrophobic residues of the shaft are given in bold. putative structural protein is located at the left end of the

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manuscript. We thank Gerhard Monreal for fruitful discussions and continuous support.

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