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Detection of herpes simplex virus mRNA in latently infected trigeminal ganglion neurons by in situ hybridization.

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Detection of Herpes Simplex Virus mRNA
in Latently Infected lrigermnal bangbon
Neurons by In Situ Hybrihation
Richard B. Tenser, M D , " t Marilyn Dawson, PhD," Steven J. Ressel, BS," and Marie E. Dunstan, MS*
Latent infection of the trigeminal ganglion with herpes simplex virus type 2 (HSV-2)was studied in guinea pigs by
in situ DNA hybridization. Frozen ganglion sections from animals killed during the period of latent virus infection
were studied under nondenaturing conditions. Some sections were treated with deoxyribonuclease (DNase) or
ribonuclease (RNase) before incubation with HSV DNA probes. HSV probes consisted of viral DNA nick translated and labeled in vitro with tritiated nucleotides. Bacteriophage lambda DNA, similarly prepared, was used as a
control probe. The lamba probe was negative in all situations, including HSV-2-infected monolayer cells in cell
culture. HSV-2 probes produced heavy label and, therefore, evidence of hybridization with HSV-2-infected
monolayer cells. When HSV-2 probes were incubated with latently infected ganglion sections, hybridization was
detected in 71% of guinea pigs and 46% of ganglia. Label was seen only in neurons, and in positive ganglia 0.3 to
595 of neurons were labeled. The amount of label was markedly decreased by pretreatment of ganglion sections
with RNase but not DNase, indicating that the DNA probes hybridized to HSV messenger RNA in the latently
infected ganglia.
Tenser RB, Dawson M, Ressel SJ, Dunstan ME: Detection of herpes simplex virus mRNA in latently
infected trigeminal ganglion neurons by in situ hybridization. Ann Neurol 11:285-291, 1982
Latent herpes simplex virus (HSV) infection of the
nervous system is common in human trigeminal and
dorsal root ganglia [ 3 , 4 , 241, and virus has also been
isolated from autonomic ganglia [ 2 3 ] . Latent H S V
infections are probably infections of neurons [5, 7,
111, and the neuronal infection is the substrate for
recurrent herpes infections. Reactivation of latent
HSV infection may be important in the pathogenesis
of H S V encephalitis, and in addition, H S V infection
may be important in the pathogenesis of some neoplastic diseases [2, 10, 121. Based o n these disease
relationships and consideration of latent H S V infection as a prototype D N A virus infection of neurons,
further understanding of HSV latency is important.
Latent sensory and autonomic ganglion infections
in experimental animals closely parallel the human
condition [5, 13, 19, 221. H S V latency has been operationally defined as an infection in which viral antigens are not detected in infected tissues and in which
infectious virus can be isolated by cocultivation but
not from homogenates of the tissues [5, 191. From
these observations it has been hypothesized that viral
DNA is present but that infectious virus particles are
not, o r possibly are present in only very small
amounts. These alternatives, which are not necessarily mutually exclusive, have been termed nonproductive and productive latent infections, respectively [ 161,
and indicate differing pathogenic mechanisms.
Recently, by the use of nucleic acid hybridization
procedures, it has become possible to study H S V latency in detail. Based o n the principle that singlestranded DNA will anneal t o complementary (in the
Watson-Crick sense) DNA that has been denatured
to single-stranded, and will also hybridize to complementary messenger R N A (mRNA), it has become
possible to investigate tissues intensively for the
presence of viral DNA and m R N A . D N A - D N A
techniques were important in the determination of
neurons as the site of latency [7]. However, this did
not provide information concerning the state of the
latent infection. More recently it was reported that
by the use of denatured radiolabeled H S V D N A ,
HSV m R N A was detected in latently infected human
ganglion tissues [ll].This finding was important because it indicated that at least partial viral transcription had occurred. The results in that study were ob-
Escueta et al: Complex Partial Seizures on CCTV-EEC 285
tained by i n situ hybridization in which t h e DNA
probe was incubated with tissue sections. Previously
it had been reported that, b y t h e more quantitative
method of solution hybridization, HSV mRNA was
n o t d e t e c t e d in ganglion tissue i n a m o u s e model of
HSV latency [14].
We performed in situ hybridization e x p e r i m e n t s i n
a guinea pig m o d e l of latent HSV infection of t h e
trigeminal ganglion t o c o m p a r e results with t h o s e rep o r t e d in humans. Evidence of HSV mRNA was observed in guinea p i g trigeminal ganglion n e u r o n s .
T h i s s u p p o r t e d t h e neuronal site of latency and t h e
presence of at least partial viral transcription. It also
indicated the value of the g u i n e a p i g model of HSV
Materials and Methods
The 333 and 186 strains of H S V type 2 (HSV-2), and KOS
strain of HSV type 1 (HSV-l), and bacteriophage lambda
CI857SuS7 were used. HSVs were plaque purified, and
stocks were grown by infection of primary rabbit kidney
cells at low multiplicities (0.1). The methods of cell culture
and virus titration have been described previously [2 11;
standard procedures were used for preparation of bacteriophage lambda [l]. Rabbit kidney cells and Vero cells
(African green monkey kidney cells; Flow Laboratories,
McLean, VA) were grown on glass coverslips and infected
at a low multiplicity for use as positive hybridization controls. These monolayers were fixed as for tissue sections to
be described.
Two groups of 12 and 13 randomly bred Hartley guinea
pigs of both sexes, weighing 200 to 250 gm, were used
(Dutchland Laboratories, Denver, PA). After corneal
scarification of anesthetized animals, 50 pl of HSV-2 strain
333 (5 x lo6 plaque-forming units/ml) was dropped onto
each eye. Guinea pigs were observed for four to seven
weeks and then were killed by exsanguination under anesthesia. No animals died acutely or showed evidence of recurrent H S V infection. In several cases, trigeminal ganglia
were removed three days after corneal inoculation to serve
as acute ganglion infection controls. Trigeminal ganglia
were removed as described previously [22] and, after
washing in cold isotonic salt solution, were frozen in O C T
(Lab-Tek Products, Naperville, IL). Ganglia were kept at
-70°C prior to cryostat sectioning. Sections (12 p ) on glass
slides were fixed in ethanol at 4°C and were kept at -70°C
until assayed.
Standard procedures were used to purify virions and
viral D N A [18]. Briefly, 1 hour after infection of
monolayer cells, [3H]-labeled thymidine (5 pCi/ml) was
added to the medium (15 ml). When viral cytopathology
was marked, cultures were freeze-thawed. After low-speed
centrifugation, cell-free supernates were stored at 4°C and
cellular debris was sonicated and treated with 1% Nonidet
P-40 (NP-40) and 1% sodium deoxycholate. These supernates were centrifuged, pooled with previous supernates,
and treated with zinc acetate (1 M) to precipitate virions
(Howett MK: personal communication, 1979). This material was resuspended in saturated ethylenediamine tetraacetic acid (EDTA). NP-40 and sodium deoxycholate in
Annals of Neurology
Vol 11 No 3
March 1982
tris(hydroxymethy1)aminomethane hydrochloride (Tris)EDTA buffer was added, and the suspension was centrifuged in a cesium chloride step gradient (1.2 and 1.4
gmlml) for 29'2 hours at 25,000 rpm in a SW27 rotor. The
partially purified virion band was removed, and sodium
dodecylsulfate (2.596) and proteinase K (10 mg/ml; E.
Merck, Darmstadt, West Germany) were added to digest
the virions. This material was then layered onto a continuous 10 to 30% sucrose gradient in SW27 tubes and centrifuged at 15,000 rpm for 16 to 18 hours. Fractions were
collected by bottom puncture, and the virus D N A peak
was determined by measurement of 3H activity in a scintillation counter. Appropriate fractions were concentrated by
dehydration (Aquacide; Calbiochem, La Jolla, CA). D N A
was purified by centrifugation (50.3 rotor) at 35,000 rpm
for 6 5 hours in a cesium chloride gradient. Fractions were
collected by bottom puncture and the virus D N A peak was
again determined. After dialysis, viral D N A was extracted
in phenol : chloroform : isopropanol (25 : 25 : 1) and then
precipitated in 3 M sodium acetate in isopropanol. Purified
D N A was washed with 70% ethanol and stored in TrisEDTA buffer at -20°C before nick translation.
Nick translation methodology was used to label viral
D N A probes in vitro: Single-stranded cuts (nicks) were
randomly produced in the double-stranded viral D N A . In
the presence of D N A polymerase, the nicks were then
filled in with 3H-labeled nucleotides to produce labeled
D N A . The procedure of Rigby et a1 [15], with slight
modification, was used. Briefly, purified viral D N A was incubated with activated deoxyribonuclease (DNase; 1
mg/ml; Worthington Biochemical, Freehold, NJ) to produce D N A nicks and then with D N A polymerase I ( 5 to 10
units; Boehringer Mannheim, Indianapolis, I N ) in the
presence of [3H]deoxycytidine triphosphate (1 mCi/ml)
and [3H]deoxythymidine triphosphate (5 mCi/ml). When
high activity was determined in reaction aliquots, the reaction was stopped by adding 0.1 M EDTA and heating the
mixture to 65°C. Labeled D N A was separated from unincorporated nucleotides by Sephadex filtration (G 50-80).
Nick-translated viral D N A probes contained approximately 20,000 cpmlpl (1 to 2 x lo7cpm/pg DNA).
T h e procedure of Brahic and Haase [6] for in situ
hybridization was utilized with slight modification.
Tissue sections o n slides and monolayer cells were
first treated to enhance penetration of the viral D N A
probes and then were incubated with the 3H-labeled
probes. Target tissues were treated with 0.2 M hydrochloric acid at room temperature, with sodium
chloride (0.3 M)-sodium citrate (0.03 M) buffer (SSC) at
70°C, and with proteinase K (1 ,ug/ml) at 37°C. In some
instances, tissue sections were then treated with either
DNase (5 pglml) or RNase 1-A (100 pg/ml; Sigma
Biochemical, St. Louis, MO). RNase was heated to 90°C
for 10 minutes to destroy any DNase present. DNase preparations assayed by the Kunitz procedure had appropriate
enzyme activity.
Target tissues were dehydrated in ethanol and dried.
Volumes of 3H-labeled probes containing 40,000 to 50,000
cpm per coverslip (two-ganglion section) and 1 pg of calf
thymus D N A were boiled for 10 minutes to denature the
double-stranded viral D N A into single-stranded D N A .
D N A was rapidly cooled, and 10 pl of probe mixture con-
taining 50% formamide and 3~ SSC buffer was added to
targets. Coverslips were applied and sealed with rubber
cement, and tissues were incubated at 47°C for 48 to 60
hours. Coverslips were removed and tissues were
thoroughly washed and then dehydrated in ethanol. After
being dried, slides were dipped into photographic emulsion
(NTB-2; Kodak Corp, Rochester, NY) and exposed at 4°C
for three to four weeks. Slides were then developed (Rodinal developer; Agfa-Gavaert Inc., Teterboro, NJ; Kodak
fixer) and air dried, and were stained with 4% Giemsa;
after addition of a drop of immersion oil, coverslips were
applied. Tissue monolayer cells were examined at 100 to
4 0 0 for
~ the presence of silver grains indicating hybridization of the 3H-labeled probes with complementary nucleic
HSV Infected Monolayer Cells
The specificity of the in situ hybridization reaction
was demonstrated by comparing the reaction obtained with HSV and lambda probes on lytically infected monolayer cells. In areas of HSV-2
cytopathology, HSV- 1 and HSV-2 probes produced
heavy labeling (Fig 1A) whereas results with lambda
probes were negative (Fig 1B). Label with the 333
and 186 HSV-2 probes could not be differentiated.
Label with HSV-1 probes was similar to that with
HSV-2, although the HSV-2 probes produced a
heavier label in the HSV-2-infected cells than did
HSV-1. This difference was studied further by blind
reading of pairs of Vero cell monolayer cultures on
coverslips infected with HSV-2 and incubating with
HSV-1 and HSV-2 probes. The homologous HSV-2
system was identified by the presence of heavier label
in 25 of 34 pairs (p = 0.01). For this reason, HSV-2
probes were used for subsequent study of tissue sections.
Acate Trigeminal Ganglion Infection
Trigeminal ganglia removed three days after corneal
inoculation with HSV-2 were used as an additional
control; we previously detected infectious HSV in
100% of ganglia from similarly infected guinea pigs
([22] and unpublished data). In acutely infected
ganglion tissue, evidence of hybridization was easily
found. Label was present in neurons and was also observed in supporting cells (Fig 2). The specificity of
the hybridization reaction was indicated by the sparse
concentration of background grains in uninfected
areas. Hybridization was not detected with the
lambda D N A probe. As an additional control, tissue
sections of four trigeminal ganglia from 2 uninfected
guinea pigs were studied with the HSV probe; no
labeled cells were seen.
Latent Trigeminal Ganglion Infection
Ganglion sections from animals killed during the period of patent infection were examined for the pres-
F ig I . Areas of HSV-2 cytopathology i n Vero cell monolayers.
(A) Incubated with nick-translated HSV-2 D N A probe.
Heavy label of silver grains indicates hybridization. Uninfected adjacent area does not show hybridization label. ( B )
Incubated with nick-translated bacteriophage lambda DNA.
Lack of label indicates lack of hybridization and, therefore,
specificity of the reaction in A. (Giemsa; x800 before 20% reduction.)
ence of clusters of silver grains. At least two sections
from each ganglion were studied with the HSV-2
probes. Cell morphology was variable, but in most
sections preservation was good and neurons were
easily recognizable. Large amounts of label in even a
single cell were considered to indicate that the
ganglion was positive for hybridization. Such hybridization as well as good tissue preservation are
shown in Figure 3. Several positive neurons are seen
at high magnification in Figure 4. A neuron with less
label than that in Figure 4C was scored as 5 and by
itself was not taken to indicate evidence of hybridization in a ganglion section.
Hybridization was detected in ganglia of 15 of 2 1
guinea pigs (71%) and in 19 of 41 ganglia studied
(46%) (Table). These percenrages are somewhat
greater than virus isolation results obtained by cocultivation, in which we found latent HSV-2 infection in
Tenser et al: Latent Herpes Simplex Virus Infection
44% of guinea pigs and 33% of ganglia (unpublished
data). In hybridization-positive ganglia, 0.3 to 5% of
neurons were labeled; evidence of label was not detected in nonneuronal cells. Duplicate sections of all
ganglia were tested with lambda probes and did not
show evidence of hybridization.
Latent Trigeminal Ganglion Infection after Treatment
with DNase and RNase
By our standard tissue preparation procedure, D N A
in ganglion sections was not denatured, and probes
were thought to have hybridized with HSV mRNA.
This was investigated further by treating ganglion
sections with DNase and RNase before incubation
Annals of Neurology
Vol 11 No 3
March 1982
Fig 2. Trigeminal gunglion section from acutely infected
guinea pig (three days after corneal inoculation). (A)Group of
infected neurons and supporting cell.s. Speci$city of-thehybridization reaction is indicated by the presence of only f&
background silver grain.[. ( B ) Clusters of .silver grains over u
satellite cell (arrow) arid supporting cells are eziident and contrast with the neuronal localization observed during lutency.
(Giemsa: ~ 8 0 0 . 1
F i g 3 . Section of the trigeminal ganglion from latently infected
guinea pig. Lou>-powermagnification shows good tissue preservation. A single neuron with cluster of silver grains indicates hybridization (arrow). This neuron is shown at higher
magnification in the inset. (Giemsu;Jigure ~ 2 0 0inset
Detection of Herpes Simplex Virus i n Latently
Infected Guinea Pig Trigeminal Ganglion
Neurons by In Situ Hybridization a
Ganglia Positive/
Ganglia Tested
(% Positive)
19/41 (46)
HSV-2 D N A probe
0/41 (0)
lambda DNA probe
17/38 ( 4 5 )
DNase pretreatment
HSV-2 DNA probe
3/38 (8)
RNase pretreatment
HSV-2 DNA Drobe
Animals Positive/
Animals Tested
(5% Positive)
15/21 (71)
0121 ( 0 )
16/20 (80)
3/20 (15)
aGuinea pigs were inoculated with HSV-2 and sacrificed four to seven
weeks later. Nick-translated 186 and 333 probes were used for both groups
of animals, and results were indistinguishable. By chi-square testing, p values comparing HSV-2 D N A probe and DNase pretreatment results with
RNase pretreatment results were significant at 40.001.
HSV = herpes simplex virus.
with viral probes. DNase pretreatment slightly diminished the intensity of label and also decreased tissue preservation in some instances, but label was
readily detected in neurons in many sections (Fig 5).
After DNase treatment, clear evidence of hybridization was observed in 17 of 38 (45%) ganglia studied
(see the Table). With RNase pretreatment, only 3 of
38 (8%) ganglia were scored positive, and neurons in
those ganglia were much less definite that the ones
shown in Figure 5 . These results indicate that HSV
D N A probes primarily hybridized with complementary mRNA present in latently infected ganglia.
As has previously been indicated by DNA-DNA
hybridization studies of dorsal root ganglia of mice
[ 7 ] , the sites of latent HSV infection were within
ganglion neurons. However, in studies by Stevens
and his colleagues [ 7 ] ,it was necessary to incubate
ganglion tissue in vitro before hybridization because
of the presence of low concentrations of viral
genomes that could not be detected when hydridization was attempted with fresh tissue. Incubation in
vitro probably permitted replication of viral DNA,
thereby increasing the number of potential hybridization sites. The use of D N A probes to detect viral
mRNA might be expected to result in increased sensitivity since several R N A transcripts of a single
D N A molecule might be present. The presence of
multiple mRNA transcripts would increase the
number of potential binding sites for probe DNA.
F i g 4. Trigeminal ganglion sections from 3 other latently infected guinea pigs; neurons show evidence of hybridization. A
labeled neuron, such us i n A, B , and C , indicuted that the
ganglion was positive. (Giemsa; ~ 8 0 0 . 1
Tenser et al: Latent Herpes Simplex Virus Infection
This may explain why we were able to detect hybridization with fresh guinea pig tissues and why Galloway
et a1 [111 detected hybridization with human tissues.
It is noteworthy that a period of in vitro incubation
was not necessary.
One explanation for our results that should be discussed is the possibility that, rather than detecting
latent HSV, we detected reactivated infectious HSV.
HSV reactivates spontaneously in humans and rabbits and may result in asymptomatic virus shedding o r
in symptomatic disease [9, 201. Reactivation might
also occur spontaneously in guinea pigs. Some investigators have reported reactivation of HSV-2 in
guinea pigs after vaginal and footpad inoculation, and
also described the presence of infectious virus in
nonneuronal tissues during latency [8, 171. However, we did not isolate infectious virus during latency from footpad tissue (0 of 38 footpads from 19
animals), from vaginal swabs (0 of 7 5 from 10 animals), or from ocular swabs (0 of 126 from 18 animals) after appropriate inoculation of guinea pigs
(unpublished data). In addition, we did not isolate
cell-free infectious virus from trigeminal ganglion
homogenates (0 of 20 ganglia from 10 animals) of
latently infected guinea pigs (unpublished data).
Based on these observations and the high percentage
of hybridization-positive animals, it is unlikely that
hybridization detected reactivated replicating HSV,
although this cannot be completely excluded.
Evidence of hybridization was detected using
ganglion tissue sections that had not been subjected
to denaturing conditions. Therefore, labeling was
consistent with the viral probe hybridizing with viral
mRNA rather than with viral DNA. This possibility
was further investigated by the use of nucleases before hybridization to destroy R N A and DNA. Treatment of tissue sections with DNase resulted in
minimally decreased hybridization, whereas RNase
treatment greatly decreased hybridization. This
result strongly indicated that the viral D N A probe
hybridized with the viral mRNA present.
Although evidence of HSV-specific R N A was detected in this study and in that reported by Galloway
et a1 [ l l ] ,viral R N A was not detected in a solution
hybridization study with latently infected ganglia
from mice [ 141. While solution hybridization provides results that are more quantitative than those
produced by in situ hybridization, the latter is probably more sensitive when the concentration of nucleic acid is small and, more importantly, is localized
F i g 5 . Trigeminal ganglion sections from 3 latently infected
gutrzea pigs pretreated with DNuse before the hybridization
reaction (A-C). Label in neurons indicates hybridization
ofprobe D N A with HSV mRNA. (Giemsa; X800.j
290 Annals of Neurology
Vol 11 No 3 March 1982
to few cells. The in situ procedure, however, which is
dependent on tissue sections, is more subject to
sampling difficulties. With reference to sensitivity,
it is worthwhile noting that while Galloway and coworkers reported that their in situ results were similar whether HSV-1 or HSV-2 D N A was used as the
probe [ 111, we observed maximum labeling when
both infecting and probe virus were homologous
It is possible that the hybridization procedures
detected neuronal transcription of the entire viral
D N A o r that only partial transcription had occurred.
If partial transcription occurred, the same viral information might not be transcribed in all infected
neurons. The degree of transcription and questions
relating to the portion (or portions) of the viral
genome transcribed can be approached using probes
consisting of viral D N A that has been cut with appropriate restriction endonucleases. In this way, only
mRNA transcribed in neurons from specific D N A
regions complementary to the particular probe
D N A restriction fragment(s) used would be detected. Using such an approach and utilizing the
D N A fragment coding for viral-specific thymidine
kinase, evidence of transcription of at least this region of viral D N A was detected (McDougall JK: personal communication, 1980). HSV-specific thymidine
kinase is of particular interest because Yamamoto et
a1 [25] reported its presence in latently infected
ganglia, and we have provided evidence that its expression is important for infection of neurons [21].
Additional probe D N A fragments of HSV might
provide further understanding of HSV latency and
latent infections of the nervous systems.
Supported by Teacher Investigator Award NS 00248 and Grant NS
14568 from the NINCDS and by Grants CA 27503 and CA 18450
from the National Cancer Institute.
Helpful discussions with Dr Richard Hyman are gratefully acknowledged, as is the editorial assistance of Melissa Reese and the secretarial assistance of Kelly Yingst and Julie Bomgardner.
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Tenser et al: Latent Herpes Simplex Virus Infection
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