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Detection of varicella-zoster virus nucleic acid in neurons of normal human thoracic ganglia.

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Detection of Varicella-Zoster
Virus Nucleic Acid in Neurons of Normal
Human Thoracic Gangha
Donald H. Gilden, MD,* Yaacov Rozenman, MD,* Ronald Murray, MD," Mary Devlin, BA,"
and Abbas Vafai, PhDV
Tissue sections from four normal human thoracic ganglia were hybridized in situ with a varicella-zoster virus-RNA
probe. Varicella-zoster virus was detected in two of four ganglia, localized exclusively in neurons. The detection of
latent varicella-zoster virus genetic material in thoracic ganglia provides further evidence of varicella-zoster virus
latency at multiple levels of the human neuraxis and supports the notion that the neuron is the primary site of
herpesvirus latency.
Gilden DH, Rozenman Y , Murray R, Devlin M, Vafai A: Detection of varicella-zoster virus nucleic acid in
neurons of normal human thoracic ganglia. Ann Neurol 22:377-380, 1987
Varicella-zoster virus (VZV) causes chickenpox (varicella) in children and shingles (zoster) in adults. Zoster
usually occurs in an area supplied by 1 to 3 contiguous
sensory ganglia. Pathological changes in dorsal root
ganglia corresponding to the dermatomal distribution of rash in zoster were first described by von
Barensprung in 1863 [14}. Ganglionic lesions were
later described at all levels of the human neuraxis
[S}, and a definitive pathological picture emerged of
an acute ganglionitis, characterized by inflammatory
hemorrhagic necrosis [ 5 } . Direct evidence of viral involvement in acutely infected ganglia was demonstrated by intranuclear inclusions in dorsal root ganglion cells [ 2 } , the detection in ganglion cells of VZV
antigen by immunofluorescence and of herpesvirus
particles by electron microscopy {6}, and the isolation
of VZV from ganglion cells {l}of humans who died
with disseminated zoster.
After an individual experiences primary infection
with VZV and the resultant ganglionitis (chickenpox),
VZV is presumed to persist in ganglia, reactivating
decades later to produce zoster. Nucleic acid hybridization has been used to demonstrate VZV in normal
trigeminal ganglia of humans without evidence of recent VZV infection (7, lo}; yet, despite the high frequency of thoracic zoster (91, VZV-DNA has not
been detected in normal human thoracic ganglia. In
part, this is due to the small size of thoracic ganglia (825 mg) and the limited quantity of DNA (10-50 wg)
that can be extracted from an individual thoracic gan-
glion for hybridization studies. To circumvent this
problem, we used the technique of in situ hybridization to search for VZV nucleic acid in normal human
thoracic ganglia.
From the Departments of *Neurology and ?Microbiology and Immunology, University of Colorado School of Medicine, Denver,
Address correspondence to Dr Gilden, Unlversity of Colorado
School of Medicine, Department of Neurology, Box B-182, 4200
E Ninth Ave, Denver, C O 80262.
co.
Material and Methods
Human GangLia
A 5 1-year-old man died a sudden death from drowning. An
autopsy 23 hours later revealed no skin lesions characteristic
of recent chickenpox or shingles. Four thoracic ganglia were
aseptically removed by the anterior approach, the nerve
roots were trimmed from the ganglia, and the tissue was
immediately fixed and processed for in situ hybridization.
Preparation and Specificity of the VZV-RNA Probe
The VZV SalI-P DNA fragment was cloned into an in vitro
transcription vector plasmid (pSP65) as described [13]. The
recombinant DNA was cleaved downstream from the DNA
ihsert, and the linearized DNA was used as a template for
RNA transcription. 32P-labeledRNA was transcribed from
one strand of VZV SalI-P as described { 131, using SP6 RNA
polymerase. The specificity of the "P-labeled FWA probe
was determined by hybridization to DNA from the human
herpesviruses (herpes simplex virus, cytomegalovirus, and
Epstein-Barr virus) and to DNA of human liver, BSC-1
monkey kidney cells, and salmon sperm.
In situ Hybridization
Microscope slides were cleaned in 1 M HCI; washed in
deionized water; placed in 95% ethanol; wiped with lens
paper; air-dried; incubated for 3 hours at 65°C in a solution
of 0.02% polyvinylpyrrolidone, 0.02% Ficoll, and 0.02%
Received Dec 8, 1986. Accepted for publication Feb 9, 1987.
377
bovine serum albumin in 3X SSC (1X SSC = 0.15 M NaC1,
0.015 M trisodium citrate, p H 7.4); followed by a rinse in
deionized water and fixation in a solution of 3 parts 95%
ethanol to 1 part glacial acetic acid for 20 minutes at room
temperature. Ganglia were fixed at 4°C overnight in 0.1 M
lysine monohydrochloride in 0.05 M Na2HP04, containing- I
0.5% paraformaldehyde (PLP) and 1.3 mM sodium metaperiodate. The tissues were then dehydrated in graded concentrations of ethanol, incubated twice in chloroform, and
embedded in paraffin. Sections (4-6 +) were placed on PLPtreated microscopic slides with 0.1% Elmer's glue. The slides
were deparaffined in xylene, rinsed with 959% ethanol, airdried, baked at 80°C for 2 hours, and stored at room temperature. Prior to in situ hybridization, the slides were rehydrated, washed for 20 minutes at room temperature in 0.2 M
HCI, rinsed in deionized water, and incubated for 15 minutes at 37°C in 10 mM Tris (pH 7.4) and 2 mM CaC12,
containing 1 wg/ml Proteinase K. Slides were acetylated
by incubating them for 10 minutes at room temperature in
0.1 M triethanolamine HCI containing 0.25% (V/V) acetic
anhydride. After dehydration, the slides were denatured for
30 minutes at 65°C in 95% deionized Formamide in 0.1X
SSC and quenched for 20 minutes in ice-cold 0.1X SSC.
The slices were prehybridized for 2 hours at 37°C in a
solution containing 50% deionized Formamide, 10% dextran sulfate, 50 mM NaHzP04 (pH 7.9), 0.8 M NaCl, 1 mM
EDTA, 0.1%) sodium dodecyl sulfate, 0.05% bovine serum
albumin, 0.05% Ficoll, 0.05% polyvinylpyrrolidone, 250
r@ml of denatured salmon sperm DNA, and 500 +g/ml of
yeast RNA. Each slice was hybridized in a fresh solution of
the above buffer, which contained 5 x lo4 to 2 x lo5 cpm
of 32P-labeled RNA, transcribed from the SalI-P VZVDNA fragment at a concentration of 1.5 x lo8 c p d k g of
DNA for 24 hours at 37"C, rinsed twice in a solution containing 50 mM NaCI, 20 mM NaH2P04 (pH 6.5), and 1 mM
EDTA, followed by two incubations in the same solution at
37°C for 15 minutes; the slide was then rinsed with 2X SSC,
and dehydrated in 70% ethanol and in 95% ethanol (each
solution also contained 0.3 M ammonium acetate). Dried
slides were dipped in a 1:1 solution of Kodak NTB-2 nuclear
track emulsion and 0.6 M ammonium acetate and stored at
4°C. Slides were developed after 1 to 6 weeks, stained with
H & E, and observed by light microscopy for the presence of
silver grains.
Results
The VZV SalI-P DNA fragment (1.75 kb) was cloned
into an in vitro transcription plasmid vector, pSP65
(Fig 1). Agarose gel electrophoresis revealed a fullsized RNA fragment (1.75 kb), which was transcribed
from one strand of SalI-P, using SP6 RNA polymerase
(Fig 2). To determine the location of SalI-P on the
VZV-DNA physical map, 32P-RNA was transcribed
from one strand of SalI-P and hybridized to SalIcleaved VZV-DNA fragments as described [ 131. The
results (not shown) indicated that 32P-RNA hybridized
to the VZV SalI-P DNA fragment that lies within the
inverted repeat sequences (IRsA'Rs) of the VZV
kb
-I------
\
'P
2?
UL
B
F
5P
4?
3?
IRs
Us
S Q P
I
TRs
K PQSR
T /-
Xmnl
h
PVJl
pSP65
"i
Y
Bag I
Fig I . Physical map of the Sall-cleaved varicella-zoster virus
(VZV)genome (top) 14). The VZV SalI-P DNA fragment was
cloned in an in vitro transcription vector system (pSP65) as previously described (bottom) { I 31. The recombinant D N A was
cleaved with Hind111 endonuclease downstreamfrom the D N A
insert, and the linearized D N A was used as a template for in
vitm transcrzption using SP6 R N A polymerase.
genome {4}. Specificity of the RNA probe was demonstrated by its ability to hybridize only to the VZV
SalI-P DNA fragment and not to DNA of the other
human herpesviruses or to DNA of human liver, monkey kidney, or salmon sperm (Fig 3). For the detection
of VZV nucleic acid in thoracic ganglia, the VZVspecific probe (32P-labeled SalI-P RNA) was hybridized in situ to four thoracic ganglia. VZV-specific
nucleic acids were detected in two of the four thoracic
ganglia (Fig 4). After 6 weeks of exposure, the grains
were localized over VZV-positive neurons in the two
positive ganglia (see Fig 4D). VZV sequences were
found only in neurons and most, but not all neurons in
the positive ganglia contained VZV (see Fig 4D).
Discussion
Until now, latent VZV had been detected only in normal human trigeminal ganglia by Southern blotting 171
or by in situ hybridization {lo}. We detected latent
VZV nucleic acid in normal human thoracic ganglia by
in situ hybridization using a VZV-specific RNA probe
378 Annals of Neurology Vol 22 N o 3 September 1987
Fig 2. Agarose gel electrophoresis of the linearized plasmid carrying the varicella-zoster virus Sall-P D N A fragment and the
R N A transcribedfrom SalI-P D N A . R N A was transcribed
from the Sall-P template and run on a 1% agarose gel as previousb described { I 3) (Lane 1 = befire deoxyribonuclease
{DNase}; Lane 2 = after digestion of D N A with DNasej.
R N A transcribedfrom Sall-P D N A was also run on a 1 %
agarose gel containing methylmercuric hydroxide (Lane 3).
transcribed from the VZV-DNA SalI-P fragment.
Specificity of the VZV probe was demonstrated by its
ability to hybridize only to VZV-DNA and not to
DNA from human tissue, monkey kidney cells,
salmon sperm, or other human herpesviruses. Two of
four thoracic ganglia obtained from a drowning victim
contained VZV nucleic acid sequences. VZV was
found only in neurons and not in satellite cells.
The detection of VZV exclusively in neurons is consistent with the previous findings of VZV in neurons
of human trigeminal ganglia [lo] and with earlier reports that neurons in both dorsal root ganglia and the
central nervous system are the site of herpesvirus latency {3, 111. Unlike trigeminal ganglia with latent
VZV infection, which reportedly contain only a few
positive neurons by in situ hybridization [101, thoracic
ganglia revealed VZV nucleic acid in the majority of
Fig 3. Specifcity of the 32P-RNA transcribedfrom the vuricellazoster virus N Z V ) Sall-P D N A fragment. D N A from herpes
simplex virus (HSV) (1.O pg9, Epstein-Baw virus (EBV)-infectedB cells (10 pd, cytomegalwirus (CMV) (1.0 pd, human
liver cells (20 pgl, BSC-1 (20 pg9 cells (in which VZV is propagated), salmon sperm ( S S ) (20&. and plasmid containing
V Z V Sail-P D N A (1.75 kb) were cleaved with SaN endonuclease, run on a 0.6% agarose gel, blot-transfewed onto nitrocellulosefilters, and hybridized to -32P-labeledSaN-P R N A { I 3}.
There is no homology between the V Z V Sall-P D N A fragment
and D N A from the other human herpesviruses or D N A of hum a n liver, monkey kidney, or salmon sperm.
neurons. This could reflect the difference in the ganglia studied (trigeminal versus thoracic), in the different probes used, in the length of autoradiographic exposure time after hybridization, or the detection of
both viral RNA and DNA with the 32P-RNA probe.
Determination of copy number for both VZV-DNA
and RNA is currently in progress.
Finally, we cannot exclude the possibility that VZV
was reactivated in ganglia immediately after the patient
died. Although no serological data were available for
this patient, we find that titers of VZV antibody as
measured by indirect immunofluorescence generally
range from 1:8 to 1:32 for sera obtained at autopsy.
This is similar to the titers found during life in most
adults 1121, suggesting that if VZV does reactivate
hours after death, measurement of antibody is not a
useful guide to subclinical reactivation.
Gilden et al: Detection of VZV Nucleic Acid
379
Fig 4. Detection of varicelkz-zoster virus (VZV) nucleic acids in
VZV-infectedcells and in n o m l human thoracic ganglia by in
situ hybridization. Uninfeced BSC-1 cells (A)and VZV-infected cells (B) were trypsinized, cytocentrifuged onto glass slides,
$xed, and hybridized to 32P-labeledR N A probe transcribedfrom
one strand of the VZV SalI-P D N A fragment by in situ bybridization. Four thoracic ganglia obtainedfrom an individual
without a recent history of zoster were prepared for in situ by-
Supported by Public Health Service Grant (NS-11037) from the
National Institutes of Health.
We thank M. Hoffman for editorial review and C. Becker for preparing the manuscript.
References
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bridization and hybridized to the 32P-labeledSall-P RNA
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not all, neurons of the positive ganglion.
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380 Annals of Neurology Vol 22 No 3 September 1987
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