Download Detection of Bovine Herpesvirus Type 1 RNA in Trigeminal Ganglia

J. gen. Virol. (1986), 67, 2515-2520. Printed in Great Britain
2515
Key words: BHV-1/ latent infection/in situ hybrMization/transcription
Detection of Bovine Herpesvirus Type 1 RNA in Trigeminal Ganglia of
Latently Infected Rabbits by in situ Hybridization
By D. L. R O C K , * ~ W. A. H A G E M O S E R , F. A. OSORIO:~ AND D. E. R E E D §
Veterinary Medical Research Institute and Department o f Veterinary Pathology, College of
Veterinary Medicine, Iowa State University, Ames, Iowa 50011, U.S.A.
(Accepted 16
July 1986)
SUMMARY
At times after conjunctival inoculation with bovine herpesvirus type 1 (BHV-1),
representing the acute and latent phases of infection, rabbit trigeminal ganglia were
examined for the presence of BHV-1 nucleic acids by in situ hybridization using a 3Hlabelled BHV-1 DNA probe. During the acute phase of virus infection, both BHV-1
DNA and RNA were detected in ganglionic neurons and occasionally in adjacent
satellite cells. However, during the latent phase of infection only viral RNA was
detectable in involved neurons. Viral RNA appeared restricted to the nucleus of
latently infected cells and was present in varying amounts in individual cells. These
results indicate that the BHV-1 genome is transcriptionally active in ganglionic
neurons during latent infection.
Bovine herpesvirus type 1 (BHV-1), a member of the alphaherpesvirus group, is responsible
for a variety of disease conditions in cattle including rhinotracheitis, conjunctivitis,
vulvovaginitis, balanoposthitis, meningoencephalitis and fatal systemic infection (Kahrs,
1981). Like other herpesviruses, BHV-1 establishes latent infections. In all likelihood, it is this
virus property alone that is responsible for perpetuation and transmission of infection in cattle
(Snowdon, 1965; Sheffy & Davies, 1972). Latent infection of cattle with BHV-1 is characterized
by: (i) most, if not all, animals are latently infected after experimental infection (Sheffy &
Davies, 1972; Davies & Carmichael, 1973); (ii) spontaneous and sporadic shedding of virus
occurs in latently infected animals (Snowdon, 1965; Bitsch, 1973; Huck et al., 1973); (iii) virus
can be reactivated predictably from latently infected animals following the administration of
glucocorticoids (Kubin, 1969; Sheffy & Davies, 1972; Davies & Carmichael, 1973); (iv) virus
persists in sensory and autonomic nerve ganglia during viral latency (Narita et al., 1978; Homan
& Easterday, 1980, 1983; Ackermann et al., 1982). The specific virus-cell interactions
underlying establishment, maintenance and reactivation of latent BHV-1 infection are
unknown.
We have described a rabbit model for latent BHV-1 infection that is consistent with the
above-mentioned observations made for latently infected cattle (Rock & Reed, 1982). In this
report, we make use of this model system together with in situ nucleic acid hybridization to
examine latent BHV-1 infection at the molecular/cellular level. Our results indicate that the
BHV-1 genome is transcriptionally active in ganglionic neurons during the latent phase of
infection.
t Present address: Department of Veterinary Science, North Dakota State University, P.O. Box 5406, Fargo,
North Dakota 58105, U.S.A.
$ Present address: Veterinary Diagnostic Center, University of Nebraska-Lincoln, Lincoln, Nebraska 685830907, U.S.A.
§ Present address: Molecular Genetics Inc., 10320 Bren Road East, Minnetonka, Minnesota 55343, U.S.A.
0000-7241 © 1986 SGM
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Short communication
New Zealand white rabbits (2.5 to 3.0 kg) were lightly anaesthetized with methoxyflurane and
inoculated in both the right and left conjunctival sac with 1.0 × 1 0 7 p.f.u, of the Cooper strain of
BHV-1 (obtained from Dr A. Strating, National Veterinary Services Laboratory, Ames, Iowa,
U.S.A.) as previously described (Rock & Reed, 1982).
Ganglionic homogenates (10~ in MEM), prepared using Ten Broeck tissue grinders, were
assayed on bovine lung (BL) cells. Ganglia for explant culture were washed in Eagle's MEM, cut
into 1 to 5 mm pieces, and cultured in MEM supplemented with 20~ foetal bovine serum,
penicillin (100 U/ml) and streptomycin (100 ~tg/ml). Cultures were maintained for 30 to 45 days
before being discarded as negative.
Extracellular virions from BHV-1 (Cooper) and pseudorabies virus (PRV) (strain BE12,
obtained from K. B. Platt, Iowa State University) infected BL cells were purified using the
procedure of Talens & Zee (1976). DNA was prepared from purified virions by Pronase-SDS
treatment followed by phenol-chloroform extraction and two cycles of sodium iodide/ethidium
bromide gradient centrifugation (Walboomers & Schegget, 1976; PeUicer et al., 1978). Herpes
simplex virus type 1 (HSV-1) (strain F) and plasmids containing cloned B a m H I fragments of this
virus genome were obtained from B. Roizman, University of Chicago (Post et al., 1980). Plasmid
DNAs were prepared using standard techniques (Maniatis et al., 1982).
Blot hybridizations of rabbit genomic DNA were performed as described by Rock & Fraser
(1983). Blots were hybridized with a nick-translated 3Zp-labelled BHV-1 probe (2 × 107 to
4 × 107 c.p.m./blot) for 48 h at 50 °C in 50~ formamide, 10~ dextran sulphate, 4X SSC (IX
SSC is 0.15 M-NaC1, 0.015 M-sodium citrate), 0"1 M-EDTA, 0.1 ~ SDS and 250 p.g/ml sonicated
denatured salmon sperm DNA. Filters were washed once for 30 min under each of the following
conditions: 1X SSC, 0.1~ SDS at 37 °C; 1X SSC, 0.1~ SDS at 65 °C; 0-5X SSC, 0.1~ SDS at
65 °C; 0-25X SSC, 0.1 ~ SDS at 65 °C. Autoradiographic exposure was 1 to 3 days. Sensitivities
approaching 10 pg BHV-I DNA were routinely obtained.
In situ hybridization was performed essentially as described previously (Brahic & Haase,
1978; Stroop et al., 1984). At various times post-infection animals were anaesthetized and killed
by perfusing with periodate-lysine-paraformaldehyde fixative (PLP) (McLean & Nolsane,
1974). Trigeminal ganglia were dissected, immersion-fixed in PLP for 24 h and embedded in
paraffin for sectioning. Paraffin was removed from tissue sections with xylene, the sections were
rehydrated in graded ethanol solutions and then sequentially pretreated with 0.2 M-HC1 and
proteinase K as described by Brahic & Haase (1978). Slides probed for viral RNA were
dehydrated in graded ethanol solutions and stored desiccated at room temperature. Slides
probed for viral DNA were rinsed in 2X SSC and further treated with DNase-free RNase A (100
~tg/ml in 2X SSC) for 30 rain at 37 °C, refixed in 5 ~ paraformaldehyde for 30 min, dehydrated in
graded ethanol solutions and stored desiccated at room temperature. DNA within tissue sections
probed for viral DNA was denatured by heating to 65 °C in deionized formamide in 0-1X SSC
for 15 min, quenched in ice-cold 0.1X SSC and dehydrated in graded ethanol solutions prior to
hybridization. Probe DNAs used in these experiments were radiolabelled by nick translation
with 3H-labelled nucleotides as described previously (Rigby et al., 1977; Stroop et al., 1984).
Sections were hybridized with 1 ng (approx. 105 c.p.m.) of 3H-labelled probe DNA per slide for
72 h at 45 °C in 2X SSC, 45 ~ formamide, 10~ dextran sulphate, 10 mM-Tris-HC1 pH 7.4, 1 mMEDTA, 1X Denhardt's solution (0"02~o bovine serum albumin, 0-02~ polyvinylpyrrolidone,
0.02~o Ficoll) and 1.0 mg/ml rabbit brain total nucleic acids. Following hybridization, slides
were washed in 2X SSC, 45~ formamide, 10 mM-Tris-HC1 pH 7.4, 1 mM-EDTA for 3 to 4 days
at room temperature. Following dehydration in graded ethanol solutions containing 0.3 Mammonium acetate, the slides were coated with NTB-2 emulsion, exposed for 3 weeks at 4 °C,
developed and stained with haematoxylin and eosin as described by Stroop et al. (1984). All
sections were examined and scored in a masked fashion. In general, backgrounds were relatively
free of non-specific autoradiographic grains; positive ceils were considered to be those cells
exhibiting grains too numerous to count easily. Tissue culture cells lytically infected with the
various herpesviruses were included as positive controls in all in situ hybridization experiments.
Cells were infected at a multiplicity of 5 and fixed (20 min at room temperature in 75 ~ ethanol25~ glacial acetic acid) at 18 h post-infection. All viral probes used in these experiments
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Table 1. Detection of BHV-1 nucleic acids in rabbit trigeminal ganglia during acute and latent
infection by in situ hybridization*
Hybridization in no. of rabbits/total no. examined
r
Probed for:
BHV-1 DNA
BHV-1 RNA
PRV RNA
HSV-1 RNA
pRB 112
(BamHI fragment B)
pRB113
(BamHI fragment Y)
Uninfected
0/5
0/5
0/4
BHV-1
acute phase
8/9
8/9
0/6
BHV-1
latent phase
0/12
10/11
0/10
ND~
0/3
0/9
ND
0/3
0/5
* Hybridization results were from two independent experiments and were recorded at 4 to 10 days postinoculation for BHV-1 acute phase and 2 to 15 months post-inoculation for BHV-1 latent phase.
t ~o, Not done.
hybridized to tissue culture cells infected with the homologous virus under the in situ
hybridization assay conditions detailed above.
The specificity of the BHV-1 in situ hybridization described was demonstrated in the
following ways: (i) under the given hybridization conditions the BHV-1 probe failed to show
appreciable hybridization to uninfected BL cells, PRV-infected BL cells and to Southern blots of
rabbit genomic DNA (data not shown); (ii) BHV-l-specific hybridization was not detected in
sections of trigeminal ganglion taken from uninfected rabbits (Table 1, Fig. 1e); (iii)
heterologous herpesvirus probes high in 700G + C content (PRV, HSV-1) failed to hybridize to
BHV-l-infected ganglionic tissue sections (Table 1) under conditions that allowed hybridization
in an homologous system.
The acute phase of BHV-1 infection was as previously described (Rock & Reed, 1982). Virus
was present in ocular swabs taken from inoculated rabbits on day 1 post-inoculation and was
detectable for an additional 9 to 15 days. Infectious BHV-1 was present in trigeminal ganglionic
homogenates from two of three animals at 5 days post-inoculation. At this time, viral DNA and
RNA were observed in neurons and occasionally other small supporting cells of the trigeminal
ganglion. Localization of viral DNA in involved neurons was predominantly intranuclear (Fig.
1a) whereas viral RNA was routinely evident in both the nucleus and cytoplasm (Fig. 1 b). On
histological examination, many BHV-l-hybridizing cells showed evidence of early necrotic
changes including pyknotic nuclei, pale eosinophilic staining cytoplasm and irregular cell
membrane borders (Fig. I a).
During the latent phase of infection (2 to 15 months post-inoculation), BHV- 1 was detected in
explant cultures of trigeminal ganglia from 19 of 24 animals, but was not isolated from
ganglionic homogenates (N = 5). BHV-1 RNA, but not DNA, was detected in ganglionic
neurons from 10 of 11 latently infected animals (Table 1). Involved cells, making up
approximately 0-3 ~o of all neurons, were morphologically normal in appearance. In contrast to
the RNA hybridization pattern seen in acutely infected cells, latently infected neurons
contained autoradiographic grains almost exclusively over the nucleus of the cell, indicating an
apparent restriction of viral RNA to the cell nucleus (Fig. 1 c, d). In addition, it was noted that
individual latently infected neurons appeared to express varying levels of BHV-I RNA.
The above results indicate that the BHV-1 genome is transcriptionally active in latently
infected ganglionic neurons. Latency-related viral transcription has also been observed with
HSV-1 and HSV-2 in ganglia from humans and experimental animals (Tenser et al., 1981;
Galloway et al., 1982; Stroop et al., 1984). Thus, transcription from latent viral genomes appears
to be a common feature of the alphaherpesviruses.
The apparent restriction of viral RNA to the nucleus of latently infected neurons described
here with BHV-1 has also been observed with HSV-1 (Stroop et al., 1984). It seems unlikely that
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S h o r t communication
Fig. 1. Detection of BHV-1 nucleic acids in trigeminal ganglia of acutely and latently infected rabbits
by in situ hybridization. (a) BHV-l-specific DNA in a neuron of the trigeminal ganglion of a rabbit 5
days post-infection. Note that autoradiographic grains are localized in the nucleus of the cell and that
the cell shows evidence of early necrotic changes. (b) BHV-1 RNA in a ganglionic neuron during acute
phase of infection (5 days post-inoculation). Viral RNA is evident in both the nucleus and cytoplasm of
the involved cell. (c,d) Detection of BHV-l-specific RNA in ganglionic neurons from latently infected
rabbits (120 days post-inoculation). Viral RNA is localized in the nucleus of both hybridizing cells. (e)
Section of trigeminal ganglion taken from an uninfected control animal and hybridized with a BHV-Ispecific probe. Typical background levels of non-specific grains can be seen. Autoradiographic
exposure for all slides was 3 weeks.
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this restriction is absolute because available evidence from HSV-1 latency studies suggests that
at least some viral proteins are present and most likely synthesized, at least intermittently,
throughout the latent phase of infection (Yamamoto et al., 1977; Green et al., 1981). However,
this relative restriction and apparent accumulation of viral R N A in the nucleus of latently
infected neurons possibly could represent a post-transcriptional regulatory event involved in the
maintenance of this virus-cell interaction. Post-transcriptional regulation of viral gene
expression involving preferential retention of specific viral transcripts in the nucleus of cells
during the course of productive infection has been described with several of the herpesviruses,
including HSV-1 (Stringer et al., 1977; Jacquemont et al., 1980; King et al., 1980; Wathen &
Stinski, 1982).
B HV-1 was recovered from explant cultures of trigeminal ganglia taken from latently infected
rabbits, thus demonstrating the presence of the latent viral genome in this tissue. The failure to
detect viral D N A in latently infected ganglia with the in situ hybridization procedure described
here most likely represents a lack of the necessary sensitivity to detect a single or a small number
of viral genomes in a single cell. Under conditions of active R N A transcription from a single or a
small number of viral genomes it would be more likely to detect R N A than the D N A template
because of the amplification effect caused by the high R N A to template ratio present within the
cell (Moar & Jones, 1975; Copple & McDougall, 1976). Interestingly, BHV-1 D N A was easily
detected (Fig. 1 a) during the acute phase of the infection when viral D N A replication was
occurring and infectious virus was present. It would appear that amounts of viral D N A
comparable to levels seen in productively infected cells are not present in neurons during the
latent phase of the infection. This would suggest that significant replication of the viral genome
had not occurred prior to establishment of latency in these neurons. The fact that D N A ( - )
temperature-sensitive mutants of HSV- 1 appear to be capable of establishing latent infections at
the non-permissive temperature would support this contention (Gerdes et al., 1979; Watson et
al., 1980). An alternative, although less likely, explanation for this observation could involve a
qualitative difference in the viral D N A present in acutely and latently infected cells (physical
structure, degree of association with protein, etc.) that would affect the ability of the latter to
hybridize under the assay conditions used here.
The significance of viral transcription during latent BHV-1 infection is unknown. It remains
to be determined what role, if any, it plays in establishment and/or maintenance of the latent
state.
We wish to thank Dr M. S. Hofstad for his encouragement and support, Dr W. G. Stroop for helpful advice and
Ms J. Wheeler for excellent technical assistance.
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