Journal of General Virology (1992), 73, 261-268. Printed in Great Britain
261
Latent equid herpesviruses 1 and 4: detection and distinction using the
polymerase chain reaction and co-cultivation from lymphoid tissues
Hazel M. Welch, l* C. Gordon Bridges, 2 Allyson M. Lyon, 1 Lyn Griffiths I and Neii Edington I
~The Royal Veterinary College, Royal College Street, London N W 1 0 T U
1-3 Burtonhole Lane, Mill Hill, London N W 7 IAD, U.K.
and 2M R C Collaborative Centre,
The polymerase chain reaction (PCR) and co-cultivation were used to identify the lymphoreticular system
as the site of latency of equid herpesvirus I (EHV-1).
Primers for PCR were designed from aligned nucleotide sequences of the glycoprotein gB genes to amplify
the same region of both the EHV-1 and EHV-4
genomes. Subsequent restriction digests using specific
enzymes distinguished the amplified fragments of the
EHV-1 genome from those of the EHV-4 genome.
Ten weeks following an experimental infection of five
ponies with EHV-1, latent virus was detected by PCR
and recovered by co-cultivation, predominantly from
lymphoid tissues draining the respiratory tract. Significantly, latent EHV-1 also persisted in peripheral blood
leukocytes (PBL). Latent EHV-4, presumably from a
preceding natural infection, was also detected in some
tissues, including PBL, from all animals. Of additional
interest was the recovery of EHV- 1 and -4 only in the
presence of the ubiquitous EHV-2.
Introduction
documented (Burrows & Goodridge, 1984; Edington et
al., 1984; Allen & Bryans, 1986). This apparently
Latency is an important component in the pathogenesis
of the herpesviruses. It is established following an initial
infection and consequently the host remains a source for
future infections. The equid herpesviruses 1 (EHV-1,
formerly EHV-1 subtype 1) and 4 (EHV-4, formerly
EHV-1 subtype 2) are included in group A of Melnick's
classification (1971). They are classed as alphaherpesviruses on the basis of their genome structure and biological behaviour (Studdert et al., 1981; Roizman, 1982;
Honess, 1984).
Frequently, infection with either virus results in
rhinopneumonitis, although dissemination of EHV-1
may result in paresis or abortion in susceptible mares. In
addition, EHV-4 has been isolated from aborted foetal
tissue (Shimuzu et al., 1959; Studdert, 1983; Allen &
Bryans, 1986). During acute infection both viruses can be
isolated from nasal swabs. EHV-1 causes viraemia and
can be isolated from peripheral blood mononuclear ceils
(PBMCs) for up to 3 weeks post-infection (p.i.). Isolation
of EHV-1 from predominantly T lymphocytes and
monocytes during an acute infection led Scott et al.
(1983) to propose that EHV-1 is T lymphotropic and
possibly establishes latency in T lymphocytes.
Neurotropism and latency in neurons are characteristic of alphaherpesviruses (reviewed by Wildy et al.,
1982; Hill, 1985), yet failure to recover virus from the
cranial ganglia of experimentally infected horses is well
contradicted the neurological disorders associated with
EHV-1 (Manninger, 1949; Saxegaard, 1966; Jackson et
al., 1977), until it was recognized that the lesion in the
central nervous system was a vasculitis resulting from
virus replication in endothelial cells (Patel et al., 1982;
Edington et al., 1986). Clear evidence of latent EHV-I
and EHV-4 was shown by using high levels of corticosteroids (Edington et al., 1985; Browning et al., 1988).
Additionally, recovery of virus from explanted tissues
has indicated that lymphoid tissues are the sites of
latency (N. Edington, unpublished data), but this
remains to be confirmed.
Considerable similarity and gross collinearity between
the genomes of EHV-1 and -4 have been demonstrated
(Cullinane et al., 1988). Accordingly, in situ hybridization
is inappropriate for the study of EHVs, particularly for
studying naturally infected animals. Southern hybridization (Southern, 1975) is an alternative method, distinguishing viruses by their restriction pattern. Applying
this method, Efstathiou et al. (1986) successfully detected
latent herpes simplex virus type 1 in human ganglia.
However, it is insufficiently sensitive to detect latent
EHVs (H. M. Welch, unpublished data).
The polymerase chain reaction (PCR; Saiki et al.,
1985; Mullis & Faloona, 1987) has the advantages of
sensitivity and accuracy. It will detect a single copy of
target DNA in 10 ktg of DNA (equivalent to approxima-
0001-0441 © 1992 SGM
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262
H. M. Welch and others
(a)
EHV-1
BI
E
I
EHV-4
B
I
I
I
I I
EBBB
B
I
EB
F
II
I I
E E
-150 kb
.'
gB " .
•
I
rl
(b)
EHV-1
1500
.~ .
EHV-4
809
G
I
t
d b
1-'-~
D
t
V
1750
11~
~D
I) I
1059
I) H1309
2"~,-~
i
i
H
i[
2000
I D
I
t
2R
E
t
-3R
2250
• 2340
1559
1609
I
VII
I
,
vI
IV
III
Fig. 1. Collinear genomes. (a) Location of the EHV-1 and EHV-4 gB genes [from Whalley et al. (1989) and Riggio et aL (1989)
respectively]. Relative positions of gB and some restriction sites (B, BamHI; E, EcoRI) are shown. (b) Regions of aligned gB genes.
Positions of sense (~) and antisense (,--) primers are shown, as are the regions amplified. Restriction enzymes used to distinguish
EHV-1 from EHV-4 were D, DdeI; G, BglII; C, ClaI; E, BstEII; H, HhaI.
tely 1-5 x 106 diploid cells; Saiki et al., 1988) and
furthermore, the products can be subjected to analytical
manipulations (reviewed by White et al., 1989). Primers
capable of amplifying the same regions from both
EHV-1 and -4 (see Fig. 1) were designed by aligning
the nucleotide sequences of the glycoprotein gB genes of
EHV-I and -4 (Whalley et at., 1989; Riggio et al., 1989).
By designing primers from highly conserved regions, the
risk of sequence variation among natural populations
and variation resulting from laboratory adaptation was
reduced. Although 84Y/ooof the bases were identical,
sufficient base mismatches were within the recognition
sequences of restriction enzymes to allow restriction
fragment length polymorphisms (RFLPs) specific for
each virus (see Fig. 2) to be identified. In addition,
should amplification of spurious sequences occur, particularly of those from the ubiquitous EHV-2 (Kemeny &
Pearson, 1970), they would be readily identified.
In this paper we report the successful application of
PCR, RFLP analysis and co-cultivation to the study of
experimentally infected equidae.
Methods
Experimental animals
(i) Infection. Five Welsh mountain ponies from a closed herd, at 6
months of age, were infected intranasally using a swab and nebulizer as
described previously (Edington et aL, 1986). The inoculum contained
106"s TCIDs0/ml of the 1lth passage (in equine cells) of the Ab-4 (p2)
isolate of EHV-1 (Patel & Edington, 1983). Virus recovery from nasal
swabs and PBMCs was analysed daily as described previously (Patel et
al., 1982). Serum samples were taken weekly for 4 weeks and
subsequently at monthly intervals, and used to isolate complementfixing (CF) antibody (Thomson et al., 1976).
Ten weeks after the primary infection, all five ponies were killed
with intravenous barbiturate, exsanguinated, and samples of PBMCs,
retropharyngeal (RPLN), submandibular (SMLN), bronchial (BLN),
tonsillar (TON) and popliteal (PLN) lymph nodes, samples of thymus
(THY), spleen (SPL) and lung, cells from pulmonary lavage (AL) and
nasal mucosa (NM), and ganglia of the Vth cranial nerve (SLG) were
taken• These were examined for virus by conventional isolation
methods, and by co-cultivation and expIant as detailed below•
(ii) Explant and co-cultivation. These were essentially as described by
Stevens & Cook (1971). Dissected 1 cm 2 fragments of SLG, NM and
lung were adhered directly onto 25 cm 2 flasks and immediately overlaid
with equine embryonic kidney cells (EEK) or rabbit kidney (RK13)
cells which served as detectors of reactivated virus. Duplicate
preparations were made of ground tissue. Teased suspensions of lymph
nodes were seeded at 6 × 106 cells onto confluent monolayers of 25 cm 2
flasks of EEK and RK13 cells. Duplicate PBMC preparations were
made after disruption by freezing and thawing.
All preparations were kept for 10 days in RPMI 1640 medium
supplemented with 10~ foetal calf serum (FCS), after which they were
trypsinized and passaged twice if no c.p.e, was observed. Cytospin
preparations of cultures showing c.p.e, were made as described
previously (Patel et al., 1982). EHV-I, -4 and -2 were distinguished
from each other by indirect immunofluorescence (IIF) using rabbit
polyclonal antisera for screening (Thomson et al., 1976), or by using
specific monoclonal antibodies (MAbs) (Yeargen et al., 1985; Edington
et al., 1987).
(iii) Virus isolation. This was performed on EEK or RK13 cell
monolayers as described previously (Patel et al., 1982).
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Latent equid herpesviruses
P
EHV4:852
EHVI:I612
EHV4:922
EHVI:I682
EHV4:992
EHVI:I751
EHV4:I062
EHVI:I821
EHV4:II31
EHVI: 1891
EHV4:I201
EHVI:I961
EHV4:I271
EHVI:2031
EHVA:I341
EHVI:2101
EHV4:I411
EHVI:2171
G
GAAAGGATACAGCCA AC T ~ T TCCGA AGATACAATGACAG GT CC GTTTC GTGGAGGAGA
GAAAGGATACAGCCACACCT TTATCCGATAGATACAATGACAGAGTGCCAGTTTCAGTGGAGGAGA
D
"I~TCGGTCTCATCGACAGTAAGGGAAAATGTTCGTCAAAGGCCGAGTACL"I'L'~AGATAACATCATGCA
T TTC TCTCATCGA AG AA GGAAAATGTTC TC AAGGC GAGTACCTC GAGATAACAT ATGCA
TATTCACTCTCATCGATAGCAAAGGAAAATGTTCTTCTAAGGCAGAGTACCTCCGAGATAACATTATGCA
C
D
V
A
p
CCACGCGTACCACGACGACGAGGACGAGGTGGAGCTTGATTTGTG-CCGTCGAAGTTTGCAACTCq~GG
CACGC TACCACGACGACGA GACGAGGTCGAGCT GA TG
CCGTC AAGTTTGC ACTCC GGG
!CACGCTTACgACGACGACGAAGACGAGGTGGAGCTCGACCTGGTTCCGTCTAAGTTTGCTACTCCTGGC
b
A
GGCCAGAGCCTGGCAGACCACCAACGATACTACCTCTTACGTGGCGTGGATGCCATGGAGGCACT~S¢'~¢~,
G CCAGAGC TGGCA ACCAC AACGA AC ACGTCTTA GT G~ TGGATGCCATCGAGGCACTACAC
G-CCAGAGCATGGCAAACCACTAACGACACCACGTCTTATGTCGGATGGATGCCATGGAGGCACTACACA
H
TCAACGTCTGTCAACTGCATCGTCGAGGAGGTGGAG~C~GGTCCGTCTACCCCTACGACTCCTTCGCCC
TCAAC TCTGTCAACTGCAT GTCGA GAGGT GA GCGCGGTC GT TACCC TACGACTCCTT GCCC
TCAACCTCTGTCAACTGCATTGTCGAAGAG~TAGAAGCGCGGTCTGTTTACCCATACGACTCCTTTGCCC
H
P
A
D
T ~ 3 .................. ~ ........ TT ....... ~ .........................
T C ACCGGTGATATTGTGTA
TC CCGTTTTACGGCC
GG C GC GCTC
TAGA CACAA
T CGACCGGTGATATTGTGTA
CTCACCGTTTTACGGCC CGGTCAGCTGCTCAGTTAGAACACAA
H
D
TAGCTACG~-G~AGGAGCGTTTCAGGCAAGTTGAAGGGTACAGGCCCCGCGAL'~f'IJ'~ACAGTAAACTACAA
TAGCTACGC CAOGA@CG TT AG CAAGTTGAAGG TAC
CC G GACTT GACAGTAAA TACA
TAGCTACGCACAGGAGCGCTTTAGACAAGTTGAAGGATACCAACCAAGAGACTTGGACAGTAAATTACAG
p
E S
N
GCCGAAGAGL~Z~TTACCAAAAATTTTATCACTACCCCGCATGTCACCGTCAGCTCGAACTGGACCGAGA
GCCG AGAGCC GTTACCAAAAA TTTAT ACTAC CC CATGT AC GTCAGCTGGAACTGGAC GA A
GCCGGAGAGCCAGTTACCAAAAACTTTATTACTACACCTCATGTTACAGTCAGCTGGAACTGGACTGAAA
P
5
AGAAAGTCGAGGCGTGTACGCTGACCAAATGGAAAGAGU'~GAACTCGTCAgGGACGACTT~iCGG
A AA T GAGGCGTGTAC CT AC AAATGGAA GAGGT GACGAACT GTCAG GA GAGTT G GG
AAAAGATAGAGCEGTGTACACTAACTAAATGGAAGGAGGTTGACGAACTTCTCAGAGATGAGTT
GGG
EHV4:I481
GTCCTACAGATTTACT~TCGATCCATCTCGTCTACGTTTATCAGTAACACTACTCAATTTAAGTTGGAA
GTCCTACAG TTTACT TCGATCCAT TCGTC ACCTTTAT AG AACACTACTCAATTTAAG T GAA
GTCCTACAGGTTTACT TCGATCCATTTCGTCCACGTTTATTAGCAACACTACTCAATTTAAGCTAGAA
EHU4:I551
AGTGCCCCCCTTACTGAATGTGTATCCAAAGAA~CAAA
TGCCCC CT AC GA TGTGT TC AAAGAAGC AA
GATGCCCCACTCACCGACTGTGTGTCAAAAGAAGCC
AAGCCATAGACTCGATATA
A ¢CCATAGACTC ATATA
ATGCCATAGACTCTATATA
GCAGT
AAA CAGT
CAGT
Fig. 2. Aligned nucleotide sequences of EHV-1 and -4 gB genes
showing region of greatest similarity. Positions of the primers are
bracketed. []. Predicted restriction sites are indicated by A, AfiIII; B,
BanlI; C, Clal; D, DdeI; E, BstEII; G, BgllI; H, HhaI; N, NspHI; P,
Hpall; S, SalI; V, AvalII.
(iv) Tissuesfor PCR. Triplicate samples were diced, snap-frozen in
liquid nitrogen and stored at - 70 °C until required. Samples (10 ml) of
heparinized (10 units/ml) whole blood were immediately frozen until
required.
Establishment and application of the PCR
(i) Computer analysis. Computer alignment of the nucleotide
sequences of the EHV-1 and -4 gB genes was performed using
DNASTAR. The sequences were screened for restriction enzyme sites;
of 83 proposed, 11 were chosen for further consideration: AfllII, BgllI,
AvalII, BanlI, ClaI, DdeI, HhaI, HpaII, BstEII, NspHI and SalI; see
Fig. 2 for sequence alignment and position of restriction sites.
(ii) Oligonucleotideprimers. The sequences were chosen from aligned
regions showing >85% similarity (see Fig. 2), using the following
guidelines. Optimal length 20 to 25 bases; amplified frame, 200 to
800 bp; approximate GC content, 50%; non-complementary 3' ends;
minimum degeneracy at the 3' ends. Each primer pair was assessed for
primer dimer formation, secondary folding and optimum annealing
temperature using 'Olga' (Bridges, 1990). Synthesis was performed
using a Milligen Biosearch DNA Synthesizer and Short Chain
Synthesizing Kit, according to the manufacturer's recommendations.
Following cleavage, the oligonucleotides were detritylated using
NENSORB PREP columns (DuPont), lyophilized and stored at
20 °C until required.
(iii) Evaluationofprimers and restrictionsites. To assess the efficacy of
the primers and conservation of restriction sites, a number of virus
isolates were analysed. For EHV-1, the isolates were Ab-4 (P2), 9524,
-
263
W15, A183 and RACH (Patel & Edington, 1983), and Lord P isolated
from an aborted foetus and kindly provided by R. Burrows. EHV-4
isolates were 2252 and H45 (Patel et at., t983), Ky-D (I)oll & Wallace,
1954), and 198, 1511, 1999, 1363 and 1193, kindly provided by Dr J.
Mumford (The Animal Health Trust, Newmarket, U.K.). An EHV-2
isolate (G9) was recovered from the RPLN of an abattoir horse. All
isolates were grown in equine dermal (ED) (NBL-6; Flow Laboratories) or EEK cells. Cells were maintained in MEM containing Earle's
salts, supplemented with I0% FCS, 2 mM-glutamine and non-essential
amino acids in 80 cm 2 Nunc flasks (Gibco/BRL) in the presence of 5 %
CO2, at 37 °C.
(iv) Isolation of DNA. Viral DNA was extracted from infected cells
showing >85% c.p.e, according to the method of Tyros et al. (1984),
with an additional pretreatment with DNase I (20 ~g/ml) at 37 °C for
20 min.
High M r DNA was extracted from frozen tissues by standard
proteolytic digestion; approximately 80 mg of tissue was ground under
liquid nitrogen using a pestle and mortar, suspended in STE (150 mMNaC1, 10 mM-Tris-HC1 pH 8, 10 mM-EDTA) in the presence of
proteinase K (200 I~g/ml) and 0.1% SDS, incubated overnight at 37 °C,
extracted twice with phenol and once with chloroform, precipitated
twice with ethanol and taken up in TE (1 mM-Tris-HC1 pH 8, 0.1 mMEDTA) to give 500 ~g/ml. DNA from frozen blood samples (referred to
as PBL) was extracted similarly following thawing and pellet isolation
using 0-1% NP40 (BDH) (M.B. Davis, personal communication).
In addition to that from latently infected tissues, DNA was extracted
from acutely infected tissues; NM and lung acutely infected with the
EHV-1 isolate Ab-4 (P2) were obtained as described by Edington et al.
(1986). Similar tissues from a gnotobiotic foal acutely infected with the
EHV-4 isolate MD (Patel et al., 1982), were obtained essentially as
above. For use as a negative control, DNA was isolated from tissue
culture monolayers of ED cells by standard proteolytic digestion with
the addition of a pre-wash with 0.9% saline.
(v) Amplification. To facilitate handling prior to amplification, 10 gg
of high Mr DNA was digested with EcoRI, precipitated and taken up in
H20 to a final concentration of 100 ng/kd. Amplification was
performed using the Perkin-Elmer Cetus DNA Thermal Cycler Kit
and AmpliTaq Polymerase. The optimal viral DNA concentration was
determined to be 200 ng/ml and the Mg 2+ ion concentration to be
1-5 mM for each primer pair. The optimal annealing temperature for
each primer pair agreed with that predicted by Olga (see Table 1).
To reduce the risk of contamination, the following precautions were
taken. Positive displacement pipettes were used for all manipulations.
High Mr (latent) DNA was isolated in a different room from that in
which viral DNA isolation and processing of PCR products was done.
PCR reactions were set up in a laminar flow hood. AnalaR (BDH) H20
was Millipore-filtered (0-22 ~tm), autoclaved, split into aliquots and
discarded after a single use. Master mixes consisting of H20, buffer,
dNTPs and both primers were set up prior to placing aliquots of DNA
into the reaction tubes, enzyme was then added to the master mix and
dispensed into each tube. The typical reaction assay consisted of 10
mM-Tris-HCl pH 8.3, 50 mM-KCI, 1.5 mM-MgCI2, 200 gM each dNTP,
1.0 IxMeach primer, 20 ng viral DNA or approximately 2 p.g tissue DNA
and 2.1 units of enzyme. Each reaction was overlaid with 60 V.1 of
autoclaved mineral oil. All reactions were set up in groups of six, i.e.
five samples of the same tissue followed by a negative control. The
parameters for amplification of virus, acute and latent DNA were 35
cycles of denaturation at 94 °C for 1 min, annealing for 2 min at the
optimum temperature for each primer pair (see Table 1), polymerization at 72 °C for 3 min with an additional final 7 min incubation at
72 °C to complete all extensions. Reactions were held at 4 °C until
processing. PCR products were recovered by the addition of two
volumes of propan-2-ol, freezing and microeentrifugation. Pellets were
taken up in 50 p.l H20. Re-amplifications were necessary on the latently
infected tissues, and 5 ~tl of product was used for this reaction.
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264
H. M. Welch and others
Table 1. Primer details
Primer
Sequence (5'-3')t
Length
GC (~)
Tr (°C):~
1L
G G A AAG GAT ACA GCC ATA CGT C
22-mer
50
51
2L
TC[CG]§ ACC G G T GAT ATT GTG TAC
21-mer
48
47
3L
C[CG]T CTC CGT TTT ACG GCC T
19-mer
58
45
IR
CGT ACA CAA TAT CAC CGG T G G A
22-mer
50
51
2R
TAG TAA ATC TGT A G G ACC CGC G
22-mer
50
51
3R
GTA TAT CGA GTC TAT GGC TTC
21-mer
43
45
t Primer sequences were identical for EHV-1 and -4 except at those positions marked with an asterisk,
for which the EHV-1 base is given.
Tr, Optimal annealing temperature.
§ [CG], Mixed base site.
Restriction enzyme digestion and gel electrophoresis. Restriction
enzymes were purchased from Anglian Biotech or Gibco/BRL and
used according to their recommendations, with the addition of
spermidine-HC1 to 4 m~. Digests were performed on approximately
0.3 ~tg (one-twelfth) of the amplified product, electrophoresed, using a
Pharmacia mini-gel apparatus, through 4% NuSieve GTG agarose gels
(FMC BioProducts) in TBE buffer (90 mM-Tris pH 8, 90 mM-boric acid,
8 mM-EDTA, 0.5 ~tg/ml ethidium bromide) at 75 V for 1.5 to 2 h and
visualized using a 254 nm u.v. transilluminator.
Membrane hybridization. Equivocal results were analysed further by
Southern hybridization (Southern, 1975). Amplification products were
digested with DdeI, transferred from 4% NuSiev~TAE (40 mM-TrisHCI pH 8, 20 raM-sodium acetate, 1 mM-EDTA) gels onto GeneScreen
Plus and hybridized according to the manufacturer's recommendations
(DuPont). An EHV-4 gB gene probe was labelled according to the
method of Feinberg & Vogelstein (1983) and used to probe the filters
under conditions of high stringency.
Results
In vivo experimental infection
During acute infection, virus was consistently recovered
from nasal swabs between 1 and 4 days p.i., and
sporadically up to 12 days p.i. Co-cultivation of PBMCs
yielded virus from four ponies between 4 and 8 days p.i.,
with sporadic isolation at 10 and 12 days p.i. from two of
them, but only on day 11 p.i. from the fifth pony. No
virus was isolated from any sample in the third week
after infection. CF antibody titres were all less than 1 : 20
on day 1 ; 8 days p.i. three of the five showed a fourfold
increase in titre and all had a titre > 1:80 by 14 days p.i.
and thereafter.
PBMCs taken prior to infection (0 days p.i.) did not
yield EHV-1 following a single passage of co-cultivation.
Establishment of the PCR
All possible combinations of primer pairs produced the
expected fragments of identical size for each EHV-1 and
EHV-4 isolate (see Fig. 3a and b). Restriction analysis of
each fragment confirmed the predicted restriction sites
in the genomes of all EHV-1 and EHV-4 isolates with the
exception of a BanII site predicted for that of EHV-4
which was absent from all isolates (data not shown). This
could have occurred because the isolate sequenced, 1942,
was grown in non-equine cells (Cullinane & Davison,
1989) which could introduce errors. The restriction sites
were highly conserved and therefore three enzymes were
considered sufficient to distinguish EHV-1 from EHV-4
(see Fig. 1 and 3e). Single amplifications (using 1L and
1R) with modified parameters proved successful for
some tissues (data not shown). However, the greatest
precision and yield were achieved using two rounds of
amplification with nested primer pairs, the most
effective combination being 1L and 3R followed by 2L
and 2R (see Fig. 4).
Spurious amplified fragments were occasionally seen
with the primer pair 2L and 2R. They probably resulted
from secondary amplification at the start of cycling when
primers were in excess. They were more evident when
the yield was low, but did not affect interpretation of the
pattern of digested products. They were virus-specific;
indeed, the presence of EHV-1 or -4 could often be
predicted by the difference in mobility (see Fig. 4a, lanes
3 and 4).
All primer pairs were tested lbr spurious amplification
of EHV-2 and equine genomic (ED cell) D N A sequences. No amplification was seen with any combination of primers.
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Latent equid herpesviruses
(a)
l
265
(a)
2
3
4
5
6
7
L
1
(b)
L
1
2
3
4
5
6
7
8
9
10
369-246-
123-
(b)
1
2
3
4
5
6
7
2
3
4
5
6
7
8
9
369-
246 -
123-
(c)
L
1
2
3
4
5
6
Fig. 4. Amplified tissue DNA. Initial amplification utilized 1L and 3R.
Re-amplification using the primer pair 2L and 2R produced a fragment
of 294 bp. (a) A 2 ~ Nusieve gel showing a selection of positive tissues
from pony 3. Lane 1, RPLN; 2, SMLN; 3, BLN; 4, TON; 5, PBLo; 6,
PBLz8; 7, PBLT0; 8, THY; 9, AL; 10, Lung. (b) Selection of tissues
from pony 3 showing latent EHV-1 (lanes 1, 4 and 7), EHV-1 and -4
(lanes 2, 5 and 8) and EHV-4 (lanes 3, 6 and 9) digested with BstlI (lanes
1 to 3), DdeI (lanes 4 to 6) and HhaI (lanes 7 to 9). Lanes L, 123 bp
ladder.
Fig. 3. Amplified viral DNA. (a) EHV-1, (b) EHV-4. A 2 ~ Nusieve gel
showing amplification with different primer pairs: lane 1, 1L and 3R;
lane 2, 1L and 2R; lane 3, 2L and 3R; lane 4, 3L and 3R; lane 5, 1L and
1R; lane 6, 2L and 2R; lane 7, 3L and 2R. (c) Restriction enzyme
digests of EHV-1 (lanes 1, 3 and 5) and EHV-4 (lanes 2, 4 and 6) region
VI, 2L and 2R with BstEII (lanes 1 and 2), DdeI (lanes 3 and 4) and
HhaI (lanes 5 and 6). Lane L, 123 bp ladder (Gibco/BRL).
Co-cultivation and IIF
Ten weeks after infection, EHV-1 was detected by IIF on
co-cultivated explanted lymphoid tissues from four
ponies and PBMCs of two ponies (Table 2). EHV-1 was
not recovered from SLG, lung or NM, although cells
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H. M. Welch and others
Table 2. Detection of latent virus by co-cultivation of
explants 10 weeks following an experimental infection with
EHV-1
Animal
Tissue
RPLN
SMLN
BLN
TON
PLN
PBMC7o ,
SPL
THY
AL
Lung
NM
SLG
Total
1
2
3
4
5
EHV-1
EHV-2
1,2
1,2
1,2
2
2
N'r§
1,2
2
2
1,2
1,2
1,2"
2
2
NT
2
1,2
1,2
1,2"
2
1,2*
1,2*
2
1,2
1,2
1,2
2
1,2
2
1,2
1,2
2
1,2
1,2
1,2
2
2
2
2
2
2
2
2
4
2
4
2
2
2
2
5
5
5
5
5
3
5
5
3
1
2
2
3
3
1
* Presence o f EHV-4 identified by PCR.
, Presence of EHV-4 confirmed using virus-specific MAbs.
PBMCT0s PBMCs 10 weeks after infection.
§ NT, Not tested.
Table 3. Detection of latent EHV-1 and -4 by PCR and
RFLP analysis 10 weeks following an experimental infection
with EHV-1
Animal
Tissue
RPLN
SMLN
BLN
TON
PLN
PBLpil"
PBL28J~
PBLv0§
SPL
THY
AL
Lung
NM
SLG
Total
1
2
3
4
5
EHV-1
EHV-4
1
cc*
1
1
cc
1,4
1
4
1
4
1,4
1,4
1
1
1
1
1
1
1
1
1
1
cc
rcrll
4
3
3
3
2
5
3
4
5
2
3
2
2
1
1
1
1
cc
1
1
1
1
cc
1
1,4
4
1
1
1
1
1
NT
4
EHV-2 was recovered from all lymphoid tissues of all
animals and also from three lung washings (AL), two
NMs and one lung sample; it was not recovered from
SLG, or ground tissues or frozen duplicates.
4
1
i
1
1
1,4
1
1
NT
2
1
1
1
1
1
1
1
* co, Virus detected by co-cultivation but not PCR.
t PBLpi, PBMCs pre-infection.
PBL28, PBMCs 4 weeks p.i.
§ PBL70, PBMCs 10 weeks p.i.
[It,rr, Not tested.
obtained by pulmonary lavage (AL) did yield virus from
pony 2. Virus was not recovered from homogenized
tissues or lymph node suspensions that had been
subjected to freezing and thawing either. Although PCR
confirmed the presence of EHV-1 in most tissues (see
below and Table 3), it detected EHV-4 in four tissues:
two BLNs, one RPLN and one TON. Explants from
BLN and TON of pony 3 were re-passaged and tested for
EHV-4 by IIF using an EHV-4-specific MAb; the
presence of EHV-4 in both tissues was confirmed.
Amplification and RFLP analysis of tissue DNA
The PCR proved more sensitive than co-cultivation and
all ponies were shown to be latently infected with both
EHV-1 and EHV-4 (Table 3). Amplified fragments were
seen in 45 samples and subsequent digestion showed that
40 were from EHV-1 and 10 were from EHV-4; both
viruses were detected in five tissue samples, RPLN,
BLN, TON, PBL and SPL, clearly demonstrating the
efficacy of PCR and RFLP analysis (see Fig. 4). EHV-1
was detected in all tissues except NM; it was also
detected in SLG of pony 4. Amplification of EHV-4 was
more sporadic, but it was also present in the PBL (preinfection) of pony 4.
Negative amplifications were repeated at least once.
Latent virus was detected by co-cultivation, and not
PCR, in five samples: two SMLN, two THY and one
TON. Southern hybridization of these PCR products
also failed to detect virus. Weak amplifications were
confirmed using Southern hybridization.
The control for each group of amplifications remained
negative throughout all reactions.
Discussion
Latency was established in five ponies following an
experimental infection with EHV-1. Latent virus was
recovered by co-cultivation and detected by PCR
predominantly, but not exclusively, in lymphoid tissues
draining the upper respiratory tract. This supported
earlier observations (N. Edington, unpublished results),
but was in contrast to the low recovery from and
detection in tissues at the site of primary infection.
Significantly, latent virus persisted in PBL, supporting
the T lymphotropism proposal of Scott et al. (1983). The
persistence of latent virus in leukocytes allows rapid
dissemination through the vascular and lymphatic
systems should reactivation of latent virus occur.
Suitable assays performed prior to infection indicated
that the animals appeared to be immunologically naive.
Their response to antigen confirmed this (J. McCulloch,
unpublished results), despite the presence of latent EHV1 and -4. These results were not unexpected. The
transient nature of the immune response to EHV-1 and -4
is well known (Allen & Bryans, 1986; J. McCulloch, S.
Williamson & N. Edington, unpublished results). The
detection by PCR of latent virus prior to infection was
confirmed by the recovery of EHV-4 from the BLN and
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Latent equid herpesviruses
TON of pony 3. Although EHV-4 is a respiratory
pathogen, the presence of latent virus in other tissues,
including PBL, is not without precedence (Shimuzu et
al., 1959; Allen & Bryans, 1986; J. Mumford, personal
communication).
PCR and Southern hybridization failed to detect latent
virus in five tissues, from which it was recovered by cocultivation. This discrepancy is consistent with the low
prevalence of virus and the fact that analyses were
performed on duplicate samples. Southern hybridization
of these products confirmed the stringency of the
conditions employed. PCR also failed to amplify virus in
PBL collected 28 days p.i. from ponies 4 and 5. The yield
of D N A was low in these samples, such that < 5 x 104
cells were analysed.
Recovery of EHV-2 by co-cultivation was widespread.
Furthermore, growth always accompanied and preceded
growth of EHV-I and -4. A possible role for EHV-2 in
trans-activation and reactivation is suggested, particularly because trans-activation of the immediate early
gene promoter of EHV-1 by EHV-2 has been demonstrated (A. S. Purewal, A. V. Smallwood, A. Kausal, D.
Adegboye & N. Edington, unpublished results). EHV-2
also was readily recovered from most tissues of pony 5
and whereas PCR detected latent EHV-1 and -4 in this
animal, neither was recovered by co-cultivation. Host
factors must also contribute to the state of latency and
reactivation.
Latent virus in PBL of contaminating blood was
possibly responsible for the PCR detecting latent EHV-1
in the SLG of pony 4. Alternatively, EHV-1 may have
established a latent infection in SLG, but could not be
reactivated. Both EHV-1 and -4 have been detected by
PCR in a significant number of SLG taken from
naturally infected animals (H. M. Welch, unpublished
results), although they have never been recovered from
this tissue; EHV-2 has been recovered only rarely
(Edington et al., 1984).
As more data accumulate, the grouping of the family
Herpesviridae into three classes on the basis of biological
behaviour is becoming increasingly difficult. Although
neurotropism is a characteristic of alphaherpesviruses, it
is not a constraint. Pseudorabies virus (suid herpesvirus)also establishes a latent infection in PBL (Wittmann et
al., 1980; Rziha et al., 1986; Ohlinger et al., 1987) as well
as trigeminal ganglia (Sabo & Rajcani, 1976; Beran et al.,
1980; Ben-Porat et al., 1984). Conversely, Marek's
disease virus is lymphotropic and therefore is considered
by some authors to be a gammaherpesvirus; however, it
has genomic homology to alphaherpesviruses, particularly varicella-zoster virus (Buckmaster et al., 1988).
We conclude that the techniques of PCR and cocultivation together show that the alphaherpesviruses
EHV-1 and EHV-4 become latent in the lymphoreticular
267
system. This is consistent with aspects of their pathogenesis (Allen & Bryans, 1986). In situ hybridization
should identify more precisely the particular cells
involved and the specific expression of EHV-1 and
EHV-4.
We should like to acknowledge Francis Smith for technical
assistance, Miriam Dean for help with the oligonucleotide synthesis
and Anna John for the typing. This work was generously supported by
the Equine Virology Research Foundation.
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