Download Detection of two novel porcine herpesviruses with high similarity to

Journal
of General Virology (1999), 80, 971–978. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Detection of two novel porcine herpesviruses with high
similarity to gammaherpesviruses
Bernhard Ehlers, Sven Ulrich and Michael Goltz
Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany
Evidence for the existence of porcine gammaherpesviruses was obtained by PCR and sequence
analysis. Initially, samples of peripheral blood mononuclear cells (PBMC), spleens, lungs, kidneys
and livers of pigs from Germany and Spain were tested with a PCR assay which targets conserved
regions of the herpesvirus DNA polymerase gene with degenerate and deoxyinosine-substituted
primers. Amplicons of identical sequence were obtained from one spleen and two PBMC samples.
This sequence showed a high percentage of identity with the DNA polymerase genes of
herpesviruses of the oncogenic subfamily Gammaherpesvirinae. Alignment of amino acid sequences
showed the highest identity values with bovine gammaherpesviruses, namely alcelaphine
herpesvirus type 1 (68 %), ovine herpesvirus type 2 (68 %) and bovine lymphotropic herpesvirus
(67 %). Comparison with pseudorabies virus and porcine cytomegalovirus, which are the only
porcine herpesvirus species presently known, showed values of only 41 %. PCR analysis of PBMC
(n l 39) and spleen (n l 19) samples from German pigs, using primers specific for the novel
sequence, revealed a prevalence of 87 and 95 %, respectively. In this analysis, three out of eight
spleen samples from Spanish pigs were also positive. Subsequent sequencing of the amplicons
revealed the presence of two closely related gammaherpesvirus sequences, differing from each
other by 8 % at the amino acid level. The putative novel porcine herpesviruses, from which these
sequences originated, were tentatively designated porcine lymphotropic herpesvirus type 1 and
type 2 (PLHV-1 and PLHV-2). When using pig organs for xenotransplantation, the presence of
these viruses has to be considered.
Introduction
Transplantation of animal organs into humans (xenotransplantation) is considered to be a solution for the shortage
of organs presently available for allotransplantation. However,
the transfer of animal organs to humans has raised concerns
about the possibility that animal donors might harbour
microorganisms with pathogenic potential for the human
recipient and the wider population. In the past, several
pathogenic infectious agents have been transferred from nonhuman primates to men (Holmes et al., 1995 ; Gao et al., 1992).
This is one of the reasons why non-human primates are
considered to be unsuitable organ donors for xenotransplantation (Chapman et al., 1995). With pigs serving as
donors, the risk of transferring diseases to humans is supposed
Author for correspondence : Bernhard Ehlers.
Fax j49 30 4547 2598. e-mail ehlersb!rki.de
The GenBank accession numbers of the sequences reported in this
paper are AF118399 and AF118401.
0001-6056 # 1999 SGM
to be markedly lower. A close association of men with pigs has
existed over the centuries, and no serious infectious disease of
pig origin has been observed in humans, with the exception of
some influenza virus strains. Therefore, pig organs are currently
favoured for transplants (Fishman, 1994). However, in xenotransplantation, recipients of organs would receive immunosuppressive treatment in order to prevent organ rejection
(Brazelton, 1997). Under these conditions, swine pathogens
and even non-pathogenic microorganisms present in pigs
might adapt to the immunocompromised human recipient and
cause disease or recombine with human viruses to create new
pathogens. To avoid such epidemiological risks, pig breeds
have to be as free as possible of potential pathogens in order
to serve as source animals in xenotransplantation. It is therefore
necessary to identify so far undescribed, potentially pathogenic
viruses harboured by pigs in order to provide the basis for the
development of surveillance methods.
We attempted to identify novel species of the virus family
Herpesviridae because of their clinical importance during
immunosuppressive treatment after allotransplantation
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B. Ehlers, S. Ulrich and M. Goltz
(Fishman, 1997). Furthermore, although in humans and other
mammals (monkeys, horses, cattle, rodents) several different
herpesviruses had already been described (Roizman et al.,
1995), only two herpesvirus species infecting pigs were
known : pseudorabies virus (PRV ; suid herpesvirus 1) and
porcine cytomegalovirus (PCMV ; suid herpesvirus 2). PRV is
a member of the subfamily Alphaherpesvirinae. It causes
encephalomyelitis and inflammation of the respiratory tract.
Severe losses of young piglets occur in unvaccinated herds and
many non-porcine mammals are also susceptible (Wittmann &
Rziha, 1989 ; Mettenleiter, 1991). PCMV is a member of the
Betaherpesvirinae. It is found in the respiratory tract of pigs and
causes atrophic rhinitis (Ohlinger, 1989). A member of the
herpesvirus subfamily Gammaherpesvirinae infecting pigs was
not known. Therefore, we examined pigs for the presence of a
gammaherpesvirus by a modified PCR assay which targets
highly conserved amino acid motifs of the herpesvirus DNA
polymerase with degenerate and deoxyinosine-substituted
primers (VanDevanter et al., 1996).
Here, we present two new herpesvirus-related DNA
polymerase sequences that indicate the existence of two novel
members of the subfamily Gammaherpesvirinae in pigs. These
putative viruses were tentatively designated porcine lymphotropic herpesvirus type 1 and type 2 (PLHV-1 and PLHV-2).
Methods
Preparation of peripheral blood mononuclear cells and
tissue DNA. Blood samples were collected from commercial pig herds
and spleens were from a slaughterhouse in Brandenburg, Germany, in
1998. Spleens, lymph nodes, kidneys, lungs and livers from pigs of
commercial herds in Spain were kindly provided by M. Domingo
(Universidad de Barcelona, Barcelona, Spain). Total DNA of pig
peripheral blood mononuclear cells (PBMC) and of pig organs were
prepared with the QIAamp Blood kit and QIAamp Tissue kit (Qiagen).
Consensus PCR. Consensus PCR was carried out as nested PCR
with three degenerate primers (two sense primers and one antisense
primer) in first-round PCR and two degenerate primers in second-round
PCR as described by VanDevanter et al. (1996), with modifications as
described below. The first-round primers DFA (5h GAY TTY GCN AGY
YTN TAY CC 3h) (sense), ILK (5h TCC TGG ACA AGC AGC ARN YSG
CNM TNA A 3h) (sense) and KG1 (5h GTC TTG CTC ACC AGN TCN
ACN CCY TT 3h) (antisense) were mixed with the deoxyinosinesubstituted primers I-DFA (5h GAY TTY GCI AGY YTI TAY CC 3h),
I-ILK (5h TCC TGG ACA AGC AGC ARI YSG CIM TIA A 3h) and I-KG1
(5h GTC TTG CTC ACC AGI TCI ACI CCY TT 3h). Likewise, the
second-round primers TGV (5h TGT AAC TCG GTG TAY GGN TTY
ACN GGN GT 3h) (sense) and IYG (5h CAC AGA GTC CGT RTC NCC
RTA DAT 3h) (antisense) were mixed with the deoxyinosine-substituted
primers I-TGV (5h TGT AAC TCG GTG TAY GGI TTY ACI GGI GT
3h) and I-IYG (5h CAC AGA GTC CGT RTC ICC RTA IAT 3h).
PCR reactions were carried out with 50 ng PBMC or 100 ng tissue
DNA as templates. In the second round of nested PCR, 1 µl first-round
PCR reaction was used as template. Reaction mixtures (25 µl) contained
1 µM each PCR primer (Pharmacia Biotech), 200 µM each deoxynucleotide triphosphate, 0n7 U DNA polymerase AmpliTaq Gold, 2 mM
MgCl , 2n5 µl 10i GeneAmp PCR buffer II (PE Applied Biosystems) and
#
JHC
5 % DMSO (Sigma–Aldrich). For thermal cycling, Perkin-Elmer 2400
thermocyclers and 0n2 ml thin wall tube strips were used. Cycling was
performed with a time-release protocol which activates the inactive
polymerase AmpliTaq Gold only partially before cycling. Full activation
is then achieved during the first cycles (Kebelmann-Betzing et al., 1998).
In first-round PCR, the reaction mixtures were kept at 95 mC for 3 min
and then cycled 55 times with 20 s denaturation at 95 mC, 30 s annealing
at 46 mC and 30 s strand extension at 72 mC, followed by a final extension
step at 72 mC for 10 min. In second-round PCR, conditions were identical
except that the annealing and extension times were reduced to 20 s.
For sequence analysis of the second-round PCR products, 1 µl PCR
reaction was reamplified as described by VanDevanter et al. (1996).
Specific PCR. Specific primer combinations 160-S (5h CAT CTG
GTA TGC TGC CCT GTC T 3h)\160-AS (5h AAG GGT TTA TCA
ATG CTG TTT GG 3h), 167-S (5h CAG AAA GGA ATT AGC AGC
ATG TGC 3h)\167-AS (5h GAG GCA TAA AGC CAA CCT TAC AGA
3h), 170-S (5h GCT GAC CCA AAG CTC AGG ACA ATT T 3h)\170AS (5h TAT CGC CGT AGA TCA CCT TGA AGG G 3h) and 175-S
(5h GTC ACT CTA CCC TAA TCC ATC AT 3h)\175-AS (5h GCA ACA
CCA GTG AAC CCA TAC A 3h) were used for amplification of 170,
252, 277 and 284 bp of the PLHV-1 DNA polymerase, respectively.
Primer combinations 170-S\170-AS and 175-S\175-AS were also
suitable for amplification of the PLHV-2 DNA polymerase.
Primer combinations 213-S (5h TCC ATC ATG AAG ACC TGC
ATA AA 3h)\215-AS (5h CCT TAC AGA TGG AAT GGA GAT CC 3h)
and 208-S (5h CTC TTC TGT CGA ACT TGC TCA CA 3h)\212-AS
(5h ACC TTG AAG GGT TTA TCA AAC AC 3h) were designed on the
basis of the sequence differences between PLHV-1 and PLHV-2 (Fig. 3 a)
for differential amplification of PLHV-1 (393 bp) and PLHV-2 (334 bp)
fragments, respectively.
Amplification was performed after complete activation of the
polymerase for 12 min at 95 mC and then cycled 42 times with 30 s
denaturation at 95 mC, 30 s annealing at 51 mC (pair P-160), 55 mC (pair
P-170, pair P-175, P-213\215), 58 mC (P-208\212) or 62 mC (pair P-167)
and 30 s strand extension at 72 mC, followed by a final extension step at
72 mC for 10 min.
All DNA preparations from tissue and PBMC samples were tested for
the absence of PCR inhibitors by PCR with primers 145-S (5h TCT GCC
CTA TCA ACT TTC GAT GGT A 3h) and 145-AS (5h AAT TTG CGC
GCC TGC TGC CTT CCT T 3h), using the above PCR protocol at an
annealing temperature of 64 mC (amplicon, 137 bp). These primers
universally detect eukaryotic DNA (Gonzalez & Schmickel, 1986 ; Barker
et al., 1988).
Sequence analysis. PCR products were sequenced directly. PCR
primers remaining in the PCR reaction mixture were removed by
MicroSpin S-300 or S-400 HR spin columns according to the manufacturer’s instructions (Pharmacia Biotech). Sequencing of PCR products
was performed by the dideoxynucleotide termination cycle sequencing
method, using the Prism BigDye Ready Reaction Terminator Cycle
Sequencing kit (PE Applied Biosystems). Reaction and cycling conditions
were chosen according to the manufacturer’s protocol. Sequencing
reactions were run on ABI 377 or ABI 310 automated sequencers (PE
Applied Biosystems) and subsequently analysed with the Sequencing
Analysis, Sequence Navigator and AutoAssembler software packages (PE
Applied Biosystems), MacVector (Oxford Molecular Group) and the
Heidelberg Unix Sequence Analysis Resources (HUSAR) software
package (Genetics Computer Group, Wisconsin and German Cancer
Research Centre, Heidelberg).
Sequence alignments and phylogenetic analysis. Sequences
were aligned with the CLUSTAL W module of MacVector. Phylogenetic
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Detection of novel porcine gammaherpesviruses
trees were derived from amino acid sequence alignments using the
programs Protpars or Protdist and Neighbour from the PHYLIP program
package (Felsenstein, 1985). The trees were statistically evaluated using
1000 bootstrap samples.
Nucleotide sequence accession numbers. The PLHV-1 and
PLHV-2 DNA polymerase sequences were deposited in the GenBank
nucleotide sequence database (accession nos AF118399 and AF118401).
Results
Amplification of gammaherpesvirus-like DNA
polymerase sequences with degenerate and
deoxyinosine-substituted primers
A consensus PCR assay targeting regions of conserved
amino acid motifs in herpesvirus DNA polymerase genes was
used to search for novel herpesvirus species in pigs. Consensus
PCR was carried out in a nested format as described by
VanDevanter et al. (1996) who amplified the polymerase genes
of 22 known herpesviruses. Because several reports had
described the good performance of deoxyinosine-substituted
primers in targeting related (non-herpesvirus) sequences
(Rossolini et al., 1994 ; Cassol et al., 1991 ; Knoth et al., 1988),
we mixed every primer with a second deoxyinosine-substituted primer, as described in Methods, to increase the
potential of this PCR system for the detection of novel
herpesviruses. By using these primer mixtures, we had
previously amplified the DNA polymerase genes of six human,
four bovine, five equine, two porcine and two avian herpesviruses from purified viral DNA and biopsy specimens. In
addition, we had found sequence evidence for new gammaherpesviruses in PBMC of wild and zoo equids (Ehlers et al.,
1999).
In order to assay for a gammaherpesvirus in pigs, we
initially analysed samples from two distant geographic origins
with consensus PCR (primers DFA\I-DFA, ILK\I-ILK and
KG1\I-KG1 in first-round PCR ; and primers TGV\I-TGV and
IYG\I-IYG in second-round PCR). PBMC collected from eight
pigs of a herd in Brandenburg (Germany) and samples of
spleens, lymph nodes, kidneys, livers and lungs of ten pigs
originating from Spain were tested. From two German blood
samples and a spleen sample from one Spanish animal, PCR
bands of 231 bp were generated. Furthermore, amplicons of
228 bp were obtained from one spleen and three lung samples.
Sequence analysis of the 228 bp amplicons (175 bp excluding
primed regions) and subsequent FASTA analysis in the
GenBank database showed 98 % identity with the unpublished
partial DNA polymerase sequence of PCMV deposited in
GenBank by F. B. Widen, M. Banks & P. J. Lowings (accession
no. AJ222640). The PCMV origin of the sequence was
confirmed by personal communication (M. Banks, Central
Veterinary Laboratory, Addlestone, UK). The amino acid
sequences predicted from the PCMV sequence and the nearly
identical 175 bp sequence detected in this study revealed in
pairwise alignments with herpesviral DNA polymerases the
Fig. 1. Comparison of the PLHV-1 DNA polymerase sequence with the
corresponding sequence of AlHV-1. Amplification products of the PLHV-1
DNA polymerase gene were obtained from a porcine PBMC sample by
consensus PCR and semi-nested PCR, as described in the text, and
sequenced. The partial DNA polymerase sequence of AlHV-1 was taken
from the AlHV-1 sequence deposited in GenBank (accession no.
AF005370). Position 1 in this figure corresponds to position 21 269 of
the GenBank sequence. Identical nucleotides are marked with asterisks.
Black triangles mark 3h ends of primers used for amplification of PLHV-1.
The primer mixtures of first-round consensus PCR, DFA/I-DFA and
KG1/I-KG1, bind 18 bp upstream and 211 bp downstream of the PLHV-1
sequence presented ; this is indicated by the slashes.
highest similarity to members of the Betaherpesvirinae (data not
shown).
Analysis of the 231 bp sequence from spleen and PBMC
samples showed strong similarity to several members of the
subfamily Gammaherpesvirinae, with identity values of up to
64 %. Also, the amino acid sequence predicted from the 231 bp
sequence revealed a close relationship to gammaherpesvirus
DNA polymerases. Therefore, this sequence (178 bp excluding
primed regions) was likely to originate from a novel herpesvirus which was tentatively named porcine lymphotropic
herpesvirus type 1 (PLHV-1).
To extend the sequence information for PLHV-1, a seminested PCR was performed. The amplification product of PCR
with the primer mixtures DFA\I-DFA and KG1\I-KG1 was
used as a template for PCR with the sense-primer mixture
DFA\I-DFA and a PLHV-specific antisense primer (170-AS)
(Fig. 1). The sequence of the generated PCR amplicon (455 bp)
was then combined with the 178 bp PLHV-1 sequence
obtained initially. This resulted in a total sequence of 466 bp of
the PLHV-1 DNA polymerase, excluding primed regions. To
validate this sequence, PLHV-specific primer pairs (numbers
160, 167, 170 and 175) were used for generation of
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B. Ehlers, S. Ulrich and M. Goltz
Fig. 2. Multiple sequence alignment of predicted amino acids of the PLHV-1 and PLHV-2 DNA polymerases and those of other
herpesviruses. The amino acid sequences of the PLHV-1 and PLHV-2 DNA polymerases were deduced from the sequences
shown in Fig. 3 (a) and aligned with the DNA polymerase sequences of known bovine (AlHV-1, BLHV, BHV-4), ovine
(OvHV-2), human (EBV, HHV-8), monkey (HVS, RRV, RFHVMm, RFHVMn), murine (MGHV68) and equine (EHV-2)
gammaherpesviruses, obtained from GenBank (see legend to Fig. 4 for definitions and accession nos). The sequences of the
porcine alpha- and betaherpesviruses PRV (accession no. L24487) and PCMV (accession no. AJ222640) were included for
comparison. Identical amino acids are shown in bold type on a dark grey background ; similar amino acids are shown on a light
grey background.
independent overlapping PCR fragments. Sequence determination of these amplicons confirmed the sequence of PLHV-1,
which was then used for further analyses (Fig. 1).
JHE
Pairwise alignments of the predicted amino acid sequence
with herpesviral DNA polymerases revealed the highest
similarity to bovine gammaherpesvirus polymerases. Identity
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Detection of novel porcine gammaherpesviruses
(a)
(b)
Fig. 3. Comparison of partial DNA polymerase sequences of PLHV-1 and PLHV-2 and differential amplification by PCR.
(a) Partial sequences of the DNA polymerases of PLHV-1 and PLHV-2 (423 bp between the primer binding sites of 175-S and
170-AS) were aligned. Position 1 of the PLHV-1 sequence included in this figure corresponds to position 32 of the PLHV-1
sequence shown in Fig. 1. Hyphens mark positions of identity. Primer binding sites are indicated by arrows. PLHV-1-specific
primers (213-S ; 215-AS) are depicted above the PLHV-1 sequence and PLHV-2-specific primers (208-S ; 212-AS) are
indicated below the PLHV-2 sequence. Primers which amplified both sequences (175-S ; 170-S ; 170-AS) are depicted
between both sequences. (b) Analyses of three spleen samples with PLHV-specific PCR. Sample F56 is PLHV-1-positive (lanes
2, 6, 10) and sample F490 is PLHV-2-positive (lanes 3, 7, 11) as revealed by sequence analysis of amplicons obtained by
PCR with the primer combination 170-S/170-AS (see Results). Sample F54 is free of PLHV sequences (lanes 4, 8, 12). Lanes
1, 14 : Bacteriophage φx, HaeIII-digested, 500 ng. Lanes 2–5 : analysis with the primer combination 170-S/170-AS, which
amplifies both PLHV types. Lanes 6–9 : analysis with the primer combination 213-S/215-AS, specific for PLHV-1. Lanes
10–13 : analysis with the primer combination 208-S/212-AS, specific for PLHV-2.
values of 68 % were found with the corresponding sequences
of alcelaphine herpesvirus type 1 (AlHV-1) and ovine herpesvirus type 2 (OvHV-2). Comparison with the recently
described bovine lymphotropic herpesvirus (BLHV) (Rovnak et
al., 1998) revealed 67 % identity. In contrast, for the porcine
alphaherpesvirus PRV only 41 % identity was determined.
Similar low percentage values were found for the sequences of
porcine betaherpesvirus PCMV and other alpha- and betaherpesviruses ( 45 %). A multiple amino acid sequence
alignment of the PLHV-1 sequence with DNA polymerase
sequences of 12 gammaherpesviruses as well as PCMV and
PRV is presented in Fig. 2. It shows that the PLHV sequence is
gammaherpesvirus-like.
herds and 19 spleen samples randomly collected on four
different days from commercial slaughter in Brandenburg,
Germany, were analysed. Amplicons were obtained from 88 %
(37 out of 42) of the PBMC samples and 95 % (19 out of 20) of
the spleen samples. In addition, three out of eight spleen
samples of pigs originating from Spain were positive. Cell lines
of suid origin (PS, PK-15) were negative (data not shown).
Furthermore, template dilution experiments were performed
with several blood and spleen samples. In two of these
samples, at least 1000 copies of PLHV-1 DNA\100 ng total
DNA were found. PCR-negative samples remained negative
after nested PCR with the primer pairs 170 and 160. This
confirmed that the detected PLHV sequences were not part of
the pig germ line.
Survey of blood and spleen samples with specific PCR
Detection and analysis of a second
gammaherpesvirus-like sequence
The PLHV-1-specific primers 170-S and 170-AS were used
for analysis of additional porcine samples. PBMC from 42 pigs
(sows, boars, piglets) obtained from seven different commercial
The amplicons from 17 PBMC and 14 spleen samples
obtained with the primer pair 170-S\170-AS (227 bp without
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B. Ehlers, S. Ulrich and M. Goltz
the primer pair 175-S\175-AS (Figs 1 and 3 a). We analysed
two of the seven PLHV-2-positive samples and determined
identical sequences, differing from the PLHV-1 sequence.
In total, 423 bp of the PLHV-2 polymerase gene were obtained and compared with the PLHV-1 polymerase sequence.
This revealed differences at 26 nucleotide positions (Fig. 3 a).
Specific, differential amplification of PLHV-1 and
PLHV-2
In order to detect each PLHV type differentially, we
designed primer combinations targeting the sequence differences between PLHV-1 and PLHV-2. As shown in Fig. 3 (b),
the primer combination 213-S\215-AS specifically amplified
PLHV-1, whereas the primers 208-S and 212-AS were specific
for PLHV-2. Again, the amplicons were verified by sequence
analysis (data not shown). In summary, these results strongly
indicated the existence of two different porcine gammaherpesviruses.
Fig. 4. Phylogenetic tree analysis of the PLHV-1 and PLHV-2 sequence.
The phylogenetic tree was constructed with the Protpars program of the
PHYLIP program package (see Methods) using a multiple sequence
alignment of DNA polymerase sequences of PLHV-1, PLHV-2 (Fig. 3 a)
and other gammaherpesviruses, obtained from GenBank : AlHV-1
(alcelaphine herpesvirus type 1, accession no. AF005370) ; OvHV-2
(ovine herpesvirus type 2, accession no. AF031812) ; BHV-4 (bovine
herpesvirus 4, accession no. AF031811) ; BLHV (bovine lymphotropic
herpesvirus, accession no. AF031808) ; EBV (Epstein–Barr virus,
accession no. X00784) ; EHV-2 (equine herpesvirus 2, accession no.
U20824) ; HHV-8 (human herpesvirus 8, accession no. U75698) ; HVS
(herpesvirus saimiri 2 l saimiriine herpesvirus 2, accession no. X64346) ;
MGHV68 (murine gammaherpesvirus 68, accession no. U97553) ;
RFHVMm (retroperitoneal fibromatosis-associated herpesvirus of Macaca
mulatta, accession no. AF005479) ; RFHVMn (retroperitoneal
fibromatosis-associated herpesvirus of Macaca nemestrina, accession no.
AF005478) ; RRV (rhesus monkey rhadinovirus, accession no.
AF029302). Statistical analysis was performed with 1000 bootstraps. The
bootstrap values are indicated at the branches of the tree.
primer binding sites) were sequenced to prove that they
originated from PLHV-1 and not from PCMV, PRV or from
the pig. As expected, these analyses revealed the PLHV-1
sequence in most cases but, surprisingly, in some cases the
presence of a second sequence, closely related to PLHV-1, was
also observed. This sequence differed from the PLHV-1
sequence by 15 out of 227 nucleotides (7 %) (Fig. 3 a) and by six
out of 75 amino acids (8 %) (Fig. 2). Three out of 14 sequences
from spleen samples and four out of 17 sequences from PBMC
samples were of the second, diverging sequence type. The
putative virus, from which this sequence originated, was
designated PLHV-2.
The attempt to extend the sequence information for
PLHV-2 with a semi-nested PCR approach similar to that
used for amplification of PLHV-1 was not successful, probably
because of poor binding of the DFA\I-DFA mixture to the
PLHV-2 sequence. However, PLHV-2 could be amplified with
JHG
Phylogenetic analysis of PLHV-1 and PLHV-2
For phylogenetic analysis, a phylogenetic tree was constructed with the program Protpars of the PHYLIP package
and statistically evaluated using 1000 bootstrap samples. In
this analysis, the PLHV viruses clustered with the bovine
gammaherpesviruses BLHV, AlHV-1 and OvHV-2 and
showed a distance from each other that was similar to the
distance between AlHV-1 and OvHV-2 (Fig. 4). This phylogenetic tree was also supported in analyses with the programs
Protdist and Neighbour (not shown).
Discussion
The search for herpesviruses in porcine blood and tissue
samples from different geographic origins with a consensus
PCR assay targeting the herpesviral DNA polymerase with
degenerate and deoxyinosine-substituted primers indicated
the existence of porcine gammaherpesviruses. Differential PCR
and sequence analysis demonstrated the presence of two
closely related, but different gammaherpesviruses (Fig. 3). The
novel putative viruses from which these sequences originated
were tentatively named PLHV-1 and PLHV-2.
The PLHV-1 and PLHV-2 sequences differed from each
other by 26 out of 423 bases and had predicted proteins that
differed by 8 out of 141 amino acids. Fourteen of these
sequence differences were found in the region which is
amplified by consensus PCR, between the binding regions of
primers TGV and IYG (Figs 1 and 3 a). In contrast, strains of a
given herpesvirus species differ in this region by a maximum of
two bases. This was reported for strains of HHV-1, HHV-6 and
HHV-7 (VanDevanter et al., 1996) and was also found for
strains of PRV, EHV-1 and BHV-1 (Ehlers et al., 1999).
Therefore, it is likely that the PLHV-1 and PLHV-2 sequences
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Detection of novel porcine gammaherpesviruses
originated from closely related, but different virus species
rather than from two different strains of the same species.
Analysis of the partial DNA polymerase sequences of the
PLHV by amino acid sequence alignments and phylogenetic
analysis revealed a high similarity with the malignant catarrhal
fever viruses of cattle, AlHV-1 and OvHV-2, and with the
bovine lymphotropic gammaherpesvirus BLHV as shown in
Figs 2 and 4, but a considerably lower relationship to the
known porcine herpesviruses PRV and PCMV. These data
were supported by preliminary sequence data obtained by
genomic walking through the complete polymerase gene and
the immediately adjacent glycoprotein B gene of PLHV-1 (data
not shown).
The PLHV sequences were detected with the consensus
PCR assay in blood and spleen samples but not in other organs.
In additional analyses of approximately 60 porcine blood and
spleen samples with non-degenerate primers, PLHV were
detected in 80 % of the samples. From these data, we assumed
that these viruses are lymphotropic and ubiquitously present in
commercial pig herds.
So far, nothing is known about the pathogenic potential
of PLHV. The closely related bovine gammaherpesviruses
AlHV-1 and OvHV-2, as well as a more distantly related
primate gammaherpesvirus, herpesvirus saimiri, are classified
as members of the genus Rhadinovirus. They are apathogenic
in their natural hosts but cause serious lymphoproliferative
diseases in other species (Reid & Buxton, 1989 ; Fleckenstein &
Desrosiers, 1982). Accordingly, PLHV could be apathogenic
in pigs but pathogenic in other species. Alternatively, it is
possible that PLHV induces a disease in pigs that has remained
unrecognized because of the usually short lifespan (6 months)
of a commercial pig.
Even if PLHV are apathogenic in pigs, the transmission of
PLHV after transplantation of pig organs to humans could
have xenozoonotic potential, since recipients of xenografts
will be heavily and continuously immunosuppressed
(Brazelton, 1997). The viruses would be directly introduced
into the body of the recipient via an infected organ,
circumventing defence barriers like mucosal surfaces. This
could favour the transmission between pigs and humans. In
addition, PLHV viruses might recombine with the human
herpesviruses HHV-6, HHV-7 and human cytomegalovirus,
which are frequently reactivated in the post-transplantation
stage (Fishman, 1997). Therefore, virus isolation and characterization are necessary to study the biology and the
pathogenic potential of the PLHV viruses. Such data could be
the basis for evaluation of the probability of PLHV transmission to humans and the possible consequences of such
transmissions when using pig organs for xenotransplantation.
We thank A. Kluge for excellent technical assistance and H. Ellerbrok,
Robert Koch-Institut, as well as G. Letchworth, University of Wisconsin–
Madison, for critical reading of the manuscript. The supply with porcine
blood and tissue probes by M. Domingo, Universidad de Barcelona,
Spain, G. Ortmann, Staatliches Veterina$ r- und Lebensmitteluntersuchungsamt, Frankfurt\Oder, S. Burkhardt, Institut fu$ r Lebensmittel, Arzneimittel und Tierseuchen, Berlin, K.-H. Lahrmann, Klinik fu$ r Klauentierkrankheiten, Freie Universita$ t Berlin, C. Große-Siestrup, VirchowKlinikum der Charite! , Humboldt-Universita$ t, Berlin, and R. To$ njes, PaulEhrlich-Institut, Langen is gratefully acknowledged.
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