Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany1
Author for correspondence: Bernhard Ehlers. Fax +49 30 4547 2598. e-mail ehlersb{at}rki.de
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Abstract |
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Introduction |
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The goal of the present study was to characterize the genomes of the PLHVs further and to base their phylogenetic relationship and classification on analyses of complete genes. Furthermore, feral pigs (Sus scrofa) were analysed for the presence of PLHVs to find a natural reservoir.
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Methods |
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Genome walking and cloning procedures.
PLHV sequences extending from the published PLHV sequences (Ehlers et al., 1999b ) into unknown flanking regions were amplified by Genexpress GmbH (Berlin) on the basis of the Universal GenomeWalker kit from Clontech (Siebert et al., 1995
). Briefly, separate aliquots of PLHV-1- and PLHV-2-infected pig samples as well as two samples of non-infected pigs were digested with six restriction enzymes. Each batch of digested genomic DNA was then ligated separately to an adaptor. With these adaptor-ligated libraries, nested PCR was performed by using an outer adaptor primer (AP1) and an outer gene-specific primer (GSP1) in first-round PCR and the inner primers AP2 and GSP2 in second-round PCR. The amplicons obtained were ligated into the plasmid pCR-Script Amp SK(+) (Stratagene) and transformed into Epicurian Coli XL10-Gold Kan ultracompetent cells (Stratagene). Specific clones were identified by PCR with the primers AP2 and GSP2.
PCR applications and nucleotide sequence determination.
In PCR analyses of PLHV-containing feral pig samples, both PLHVs were amplified with the primer pair 170-S/170-AS. PLHV-1 was amplified selectively with the primer pairs 170-S/160-AS or 213-S/215-AS and PLHV-2 with the primer pair 208-S/212-AS. All amplified regions are located inside the DPOL gene (Ehlers et al., 1999b ). Primer pair 278-S/276-AS amplified a region of both PLHVs outside the DPOL gene (483 bp), with primer 278-S (5' GGAAATGATGCCCTTTAGGGTTTTG 3') binding to the intergenic region between DPOL and ORF A5 and primer 276-AS (5' ATGGGGCCATTCCACCTCTACTT 3') binding to the 5' part of ORF A5. The locations of all amplified regions are depicted in Fig. 2(a)
.
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The absence of PCR inhibitors in samples was tested by PCR with specificity for a conserved region of the vertebrate cytochrome B gene (Kocher et al., 1989 ).
The reactions were set up as described previously (Ehlers et al., 1999b ). Amplifications were performed after complete activation of the DNA polymerase AmpliTaq Gold (Moretti et al., 1998
) for 12 min at 95 °C and then cycled 40 times through 30 s denaturation at 95 °C, 30 s annealing at 55 °C (primers 170, 170/160, 190, 213/215, 278/276 and 280) or 58 °C (primers 208/212) and 30 s extension at 72 °C, followed by a final extension step at 72 °C for 10 min. PCR products were sequenced directly as described previously (Ehlers et al., 1999b
).
Nucleotide and protein sequence analysis.
Multiple sequence alignments were performed with the clustalW module of MacVector (version 6.01, Oxford Molecular Group). Protein pair distances were calculated with the MegAlign module of DNA* (version 3.17, DNASTAR). Phylogenetic analysis was based on a multiple amino acid alignment from which gaps or insertions unique to a particular species had been removed. The remaining conserved regions were concatenated (McGeoch et al., 1995 ) and subjected to phylogenetic tree construction by using the programs Protpars or Protdist and Neighbor from the PHYLIP program package (Felsenstein, 1985
, 1993
). The trees were evaluated statistically by using 1000 bootstrap samples.
Nucleotide sequence accession numbers.
The sequences from PLHV-1 (4643 bp), PLHV-2 (4642 bp) and PCMV (290 bp) determined in this study were deposited in the GenBank nucleotide sequence database under accession numbers AF191042 (PLHV-1), AF191043 (PLHV-2) and AF191044 (PCMV).
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Results |
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Selection of PLHV-infected pig organs for PCR-based genome walking
To select the template material for the walking approach, we tested 27 spleen and 95 blood samples for the presence of PLHV-1 and PLHV-2 with the primer pairs 213-S/215-AS and 208-S/212-AS, respectively. Samples with strong PCR reactions for one of the PLHV species were re-examined by template dilution and subsequent PCR to estimate the PLHV copy number per ng total DNA. Additionally, we attempted to exclude samples containing PCMV by the use of the PCMV-specific primer pair 199-S/199-AS. Since we had previously found PCMV [but not pseudorabies virus (PRV)] in porcine spleen samples with degenerate primers (Ehlers et al., 1999b ), we wanted to avoid a possible mixing of PLHV sequences with PCMV sequences generated in the genome walking process.
In this screening, no samples with a sufficient copy number of either PLHV-1 or PLHV-2 but negative for PCMV could be found. The reason for this was primarily the high prevalence of PCMV in the spleen samples (~90%). Blood samples had a low prevalence of PCMV (~15%) but also mostly had insufficient copy numbers of the two PLHVs. Therefore, a PLHV-1-positive spleen sample (#56) was selected that contained <100 PLHV-1 genomes per ng total DNA (<1 genome per cell) and was negative for PLHV-2. Likewise, a PLHV-2-positive spleen sample (#489) was selected that contained <10 PLHV-2 genomes per ng total DNA (<0·1 genomes per cell) and was negative for PLHV-1.
PLHV genome walking with adaptor-ligated restriction fragment libraries
The genome walking was based on a nested PCR approach, with adaptor-ligated genomic DNA fragments from the selected pig spleens #56 and #489, as described in Methods. Overlapping amplicons of 0·42 kbp were obtained, spanning a total of 5·5 kbp of PLHV-1 and 2·8 kbp of PLHV-2. The amplicons were cloned in E. coli by using the vector pCR-Script and then sequenced. Cloning and sequencing were repeated once or twice for identification of polymerase errors in individual clones. PCR was performed on the spleen samples #56 and #489 with specific primers derived from these sequences. The resulting amplicons were sequenced and compared with the walking-derived sequences. Upstream of the walking-derived region of PLHV-2, the walking process failed. Therefore, additional sequences of PLHV-2 were amplified by PCR only, by using sense primers derived from the PLHV-1 sequence and antisense primers from the PLHV-2 sequence. This approach was successful with some of the PLHV-1 primers because of the similarity of PLHV-1 and -2, shown in detail below. Consensus sequences of 4643 and 4642 bp, with 4- to 10-fold redundancy, were determined for PLHV-1 and PLHV-2, respectively, and used for further analyses. In a first step, the sequences were compared with the complete DPOL gene and adjacent downstream sequences of PCMV, since samples #56 and #489 contained not only PLHV but also PCMV. No significant stretches of identity were found in comparison with the PCMV sequences obtained from sample #489 and from the British PCMV strain B6 (M. Goltz & B. Ehlers, unpublished data) (not shown). Secondly, we analysed several regions of the DPOL genes of PLHV-1 and -2 in additional blood and spleen samples. Identical amplicons and sequences were obtained for each PLHV species, which revealed the absence of PLHV sequence variation in samples of different origin. This is demonstrated by PCR results obtained with primer pairs that detect PLHV-1 or PLHV-2 differentially (Fig. 1).
PLHV open reading frame (ORF) and nucleotide composition analyses
In an ORF analysis, the 4643 bp PLHV-1 sequence was found to span the 3' end of the glycoprotein B gene (184 bp), the complete ORF of DPOL (3012 bp) and an additional ORF (975 bp). The 4642 bp PLHV-2 sequence contained 184 bp of the 3' end of the glycoprotein B gene, the complete DPOL ORF (3003 bp) and an additional ORF of 912 bp (Fig. 2a). Both sequences exhibited very low G+C contents of 37 mol% and a marked suppression of the CpG dinucleotide frequency with a concomitant increase of TpG and CpA (Fig. 2b
). These characteristic dinucleotide frequencies have been found in several A+T-rich lymphotropic herpesviruses (Albrecht et al., 1992
; Ensser et al., 1997
; Virgin et al., 1997
).
Analysis of the DPOL genes
The DPOL ORFs of PLHV-1 and PLHV-2 (3012 and 3003 bp, respectively) differed at 194 nucleotide positions, corresponding to an identity of 93%. The deduced amino acid sequences showed 50 amino acid differences, corresponding to an identity of 95% (Fig. 3). These differences confirmed our previous suggestion (Ehlers et al., 1999b
) that the PLHVs are distinct, closely related species rather than strains of the same species. The PLHV-1 and PLHV-2 DPOLs (1004 and 1001 amino acids, respectively) were found to be the shortest herpesvirus DPOLs described so far, compared with those of other herpesviruses (10091246 amino acids). Both polymerases contained the conserved exonuclease (EXO IIII) and polymerization (motifs AC) domains of DNA-dependent DNA polymerases of eukaryotic viruses (Knopf, 1998
; Blanco et al., 1991
) (Fig. 3
).
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Analysis of feral pigs for the presence of PLHV sequences
We next investigated whether a natural reservoir for the PLHVs exists in feral pigs. For this purpose, bone marrow samples from 19 feral pigs were analysed with different PCR assays (Table 1). All bone marrow samples gave positive reactions after PCR with primers specific for both PLHVs (pair 170). Differential PCR was performed with the primer pairs 213-S/215-AS or 170-S/160-AS (PLHV-1-specific) and 208-S/212-AS (PLHV-2-specific). PLHV-2 PCR was strongly positive in 18 of 19 animals. However, PLHV-1 PCR was only positive with both primer pairs in the case of the PLHV-2-negative animal (#555). In addition, one of the PLHV-2-containing samples (#562) reacted weakly with the primer pair 170/160 (but not with the probably less-sensitive pair 213/215). Therefore, sample #562 appeared to be doubly infected (Table 1
). These results were confirmed by sequence analysis. The sequences obtained from the 18 PLHV-2- and the two PLHV-1-containing samples showed 100% identity to the DPOL genes of PLHV-2 and PLHV-1, respectively, from domestic pigs. These data suggest that the same virus species infect domestic as well as feral pigs.
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From the sequence analysis of >>700 bp of all feral PLHV isolates, we concluded that the PLHVs are infectious for domestic as well as feral pigs, with a particularly high prevalence of PLHV-2 in feral pigs.
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Discussion |
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As proposed previously (Ehlers et al., 1999b ), PLHV-1 and PLHV-2 are different species rather than merely strains of the same species, since the two viruses exhibited 50 amino acid differences in the DPOL genes. In contrast, strains of the same gammaherpesvirus species appear to contain no mutations or only a few, mostly silent, mutations in their DPOL genes, as shown for the complete DPOL genes of different strains of human herpesvirus-8 (HHV-8) (Neipel et al., 1998
), Tupaia herpesvirus (Springfeld et al., 1998
) and the recently discovered rhesus monkey rhadinovirus (RRV) (Searles et al., 1999
). The same observation was made with partial DPOL sequences of strains of several other herpesvirus species from different subfamilies (VanDevanter et al., 1996
; Ehlers et al., 1999a
). We therefore propose the taxonomic names suid herpesvirus-3 (SuHV-3) and suid herpesvirus-4 (SuHV-4) for PLHV-1 and PLHV-2.
Both PLHVs were found in organs of 19 feral pigs, with an exceptionally high prevalence of PLHV-2. No differences were found between the domestic and feral pig sequences, suggesting the presence of the same virus species in domestic and feral pigs. Therefore, transfer of these viruses between feral and domestic pig populations might be possible, as has been reported for PRV (Capua et al., 1997 ) and classical swine fever virus (Stadejek et al., 1997
). This underlines the need for the isolated breeding of pigs intended for use as organ donors in xenotransplantation.
The discovery of the porcine lymphotropic gammaherpesviruses PLHV-1 and PLHV-2 was the result of a search for unknown herpesviruses in order to unravel risk factors in xenotransplantation. Therefore, the possible pathogenic potential of the PLHVs, not only for pigs but especially for immunocompromised humans, is of concern and should be analysed further. An initial step in this direction was the comparative analysis of the genetic data acquired so far. It revealed a close relationship of the PLHVs to the ruminant gammaherpesviruses AlHV-1, on the basis of analysis of the complete DPOL gene and the A5 ORF (this study), and ovine herpesvirus-2 (OvHV-2) and bovine lymphotropic herpesvirus (BLHV), on the basis of analysis of partial DPOL sequences (Ehlers et al., 1999b ). AlHV-1 and OvHV-2 cause malignant catarrhal fever, a lymphoproliferative disease of cattle with high mortality (Reid & Buxton, 1989
), and BLHV has been discussed as a cofactor in bovine leukaemia (Rovnak et al., 1998
). This suggests possible pathogenicity of the PLHVs. However, no link has yet been discovered between the PLHVs and lymphoproliferative or neoplastic diseases. Growth of the PLHVs in cell culture is a prerequisite for such studies, and such culture experiments are currently in progress.
A common feature of gammaherpesviruses is the presence of homologues of cellular genes. They have been found or predicted to modulate the cell cycle, to regulate apoptosis, to interfere with immune functions or to function in cytokine signal transduction. These genes are probably part of the virus defence mechanisms against elimination by the host and therefore are likely to contribute to virus virulence (reviewed by Neipel et al., 1997 ; Simas & Efstathiou, 1998
). For this reason, walking on the PLHV-1 genome is continuing to find such virus virulence genes that may contribute to the pathogenicity of the PLHVs for human organ recipients in xenotransplantation.
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Acknowledgments |
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Footnotes |
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References |
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Received 19 June 1999;
accepted 26 August 1999.