1 Seal Rehabilitation and Research Centre, Hoofdstraat 94a, 9968 AG Pieterburen, The Netherlands
2 Central Laboratory, Federal State of Schleswig-Holstein, Max-Eyth-Str. 5, D-24537 Neumuenster, Germany
3 Erasmus MC, Institute of Virology, PO Box 1738, 3000 DR Rotterdam, The Netherlands
Correspondence
A. D. M. E. Osterhaus (at Erasmus MC)
osterhaus{at}viro.fgg.eur.nl
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ABSTRACT |
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Published ahead of print on 11 March 2003 as DOI 10.1099/vir.0.19005-0
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MAIN TEXT |
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The PhHV-1 isolate PB84, obtained from a fatally diseased European harbour seal (Osterhaus et al., 1985), was grown in Crandell Rees feline kidney cells (CrFK). Viral DNA was prepared from purified virions as described (Harder et al., 1996
).
A BamHI restriction fragment library of PhHV-1 PB84 DNA was established in the phagemid pBluescript SK+ (Stratagene). A 290 bp PCR fragment, generated from the PhHV-1 gD equivalent gene as described (Harder et al., 1996), was used as a probe in non-radioactive Southern blotting (ECL, Amersham) and was found to hybridize to a 9·6 kb fragment, which was sequenced to completion using nested sets of unidirectional deletion mutants and sequence-specific sequencing primers. Assembly and further analyses of the sequences, including phylogenetic studies, were achieved using the Husar clone of GCG (German Cancer Research Institute), including the PSORT II software (http://psort.ims.u-tokyo.ac.jp). The sequence of the 9·6 kb BamHI fragment has been assigned GenBank accession no. AJ290955.
The complete sequence of the 9·6 kb BamHI fragment consisted of 9578 nt. Using the FRAMES program of the GCG software, eight ORFs, each with homologues in the Us segment of other alphaherpesviruses of the genus Varicellovirus, were detected within this fragment. The order and orientation of each of the ORFs is depicted in Fig. 1. Numbering is according to the nomenclature used for herpes simplex virus type 1 (HSV-1). Inverted repeat (IR) regions of 449 bp were identified at the termini of the 9·6 kb fragment flanking the Us stretch of 8680 nt. The G+C content of the Us segment excluding the repeat elements amounts to 28 %, while the IR and direct repeat (DR) regions hold 56·5 and 44 % G+C, respectively. ORFs within the PhHV-1 Us extended into the IR elements at both sides. The central part of the Us segment is governed by 24 DRs of the sequence ATggTgTTTCATggggCgTTggg, interspersed between the ORFs encoding gG and gD.
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A negative PCR and hybridization using gD primers and probe, respectively, confirmed that residual contaminant viral DNA was not present in the RNA samples (data not shown). No DR transcripts were detected in infected cell cultures (Fig. 2). The DR probe was shown to recognize PhHV-1 DNA at the conditions described (data not shown). The ORFs encoding gG and gD were used as positive control for detection of transcription. In both PhHV-1- and FHV-infected cultures, gG and gD transcripts were detected around 1620 h p.i. (Fig. 2
).
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Alphaherpesviruses of terrestrial carnivores and PhHV-1 share significant homology at the antigenic and immunogenic levels (Osterhaus et al., 1985; Lebich et al., 1994
; Harder et al., 1996
, 1998
). Using the deduced amino acid sequences of the gB equivalents in phylogenetic analysis, CHV and FHV were identified previously as the closest relatives of PhHV-1 (Harder & Osterhaus, 1997
). Similar phylogenetic relationships were now obtained using gD. The particularly close relationship between PhHV-1, CHV and FHV is emphasized also by the genetic organization of the Us segment. This bears a remarkable resemblance between these viruses, with the exception, however, of the central DR elements detected between the ORFs of gG and gD in the PhHV-1 sequence and at the same location in the Us region of FHV, but not of CHV (Willemse et al., 1995
; Haanes & Tomlinson, 1998
). The repeated sequence stretches of FHV and PhHV-1 show some homology above background (about 46 % nucleotide identity in 150 bp).
No DR transcripts were detected using a biotin-labelled probe. However, it cannot be excluded that low level transcripts were not detected by the non-radioactive Southern blot system used in our study.
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Received 26 November 2002;
accepted 17 February 2003.