GENITAL HERPES IN ATHYMIC MICE
PROGRESSION OF INTRAVAGINAL INFECTION BY HERPES SIMPLEX-2
IN GENETICALLY ATHYMIC MICE
NORBERTO A. SANJUAN
Laboratorio de Patología
Experimental, Departamento de Microbiología, Facultad de Medicina,
Universidad de Buenos Aires
Key words: HSV-2, intravaginal infection, athymic mice,
electron microscopy, viral antigen labeling
purpose of this paper was to study the pathogenesis of wild-type
Herpes simplex-2 (HSV-2)
primary intravaginal (IVAG) infection in genetically athymic (nude)
mice. Nude (nu/nu) N: NIH(S) and Balb/c mice, as well as their
euthymic counterparts were IVAG infected with 5 x 105 pfu of HSV-2.
The progression of the infection was followed by HSV-2 immunolabeling
using the peroxidase-antiperoxidase technique in tissue sections of
the whole body, electron microscopy, and viremia titration at two
different timepoints. 70% of athymic NIH mice, 30% of euthymic NIH
mice, and 80% of both athymic and euthymic Balb/c mice developed acute
vulvovaginitis and died between 8-10 days post-infection (pi). Viremia
was not detected in either athymic or euthymic mice. HSV-2 replicated
in the vulvovaginal, vesical and perianal epithelia, then progressed
towards the central nervous system mainly along autonomic nerves and
ganglia. HSV-2 antigens were not detected in liver, spleen, kidney,
skin, heart, lung or bone marrow. The conclusion is that the T-cell
immune response seems to limit the IVAG infection of NIH mice at the
inoculation site, but is not involved in preventing HSV-2
dissemination through the blood.
de la infección intravaginal por virus Herpes simplex-2 en ratones
genéticamente atímicos. En este trabajo se estudió la patogénesis
de la infección primaria intravaginal (IVAG) por virus Herpes
simplex-2 (HSV-2) en ratones genéticamente atímicos (nude). Se
emplearon ratones nu/nu de las cepas N:NIH(S) y Balb/c. Cada animal
fue infectado IVAG con 5 x 105 ufp de una cepa salvaje de HSV-2. El
seguimiento de la infección se realizó por cortes seriados de cuerpo
entero, que fueron estudiados con peroxidasa-antiperoxidasa contra
antígenos de HSV-2, microscopia electrónica de transmisión, y
determinación de viremia a los 5 días post-infección (pi) y en
estado pre-mortem (8-10 días pi). La mortalidad por infección con
HSV-2 fue del 70% en los ratones atímicos NIH, contra el 30% de su
contraparte eutímica (p < 0.02). En la cepa Balb/c, tanto los
animales atímicos como los eutímicos tuvieron 80% de mortalidad. En
ningún caso se detectó viremia, ni la presencia de antígenos de
HSV-2 en hígado, pulmón, riñón, bazo, corazón, piel o médula
ósea. La infección progresó desde el epitelio vulvovaginal hacia el
sistema nervioso central, sobre todo a través de nervios y ganglios
pertenecientes al sistema nervioso autónomo. Se concluye que en la
cepa NIH la respuesta inmune timo-dependiente es capaz de limitar la
infección a nivel IVAG. No obstante, la ausencia de respuesta
timo-dependiente no facilita la diseminación sistémica de HSV-2.
Dirección postal: Dr. Norberto A. Sanjuan. Departamento de
Microbiología, Facultad de Medicina, UBA, Paraguay 2155, 1121 Buenos
Fax: 54-1-962-5404; E-mail: email@example.com
Recibido: 25-VI-1998 Aceptado: 12-VIII-1998
Genital herpes is one of the most prevalent human venereal
diseases, and Herpes simplex virus type-2 (HSV-2) is its principal
cause1. After a primary replication in the vulvovaginal epithelium,
HSV-2 produces a life-long latent infection in neurons belonging to
the dorsal root ganglia of the rachideal nerves. As a consequence of
reactivation of the latent virus, recurrences of lesions in the
genital tract, systemic dissemination as well as serious illness may
occur in neonates born to infected women1, 2.
Even though acyclovir and other antiviral chemicals can modify and
eventually control the recurrences of genital herpes3, vaccine
development for the prevention of HSV-2 infection is currently the
major goal4. For this to be achieved, a precise knowledge of the
immune response against HSV-2 genital infection is needed.To date, the
available information about the roles of cellular and humoral immune
responses elicited after the intravaginal (IVAG) infection by HSV-2 is
far from clear, and is often contradictory in women and experimental
models. Most animal studies focused on the IVAG cellular or humoral
immunity against HSV-2 after vaccination with attenuated strains4-7,
or recombinant viruses8-11, but little is known about the role of T or
B cell responses after the IVAG primary infection with wild type
In Balb/c mice, primary IVAG infection with wild type strains of HSV-2
produces a lethal disease12. In this model, the specific humoral
immune response does not seem to limit replication in the vaginal
epithelium13. Several attempts to study the role of cellular immunity
after HSV-2 IVAG infection in mice have been reported5, 7. Some
authors used HSV-2 strain deleted in the Thymidine kinase gene (TK-)5,
which is partially responsible for HSV-2 virulence. However, the use
of a HSV-2 strain deficient in replication does not reveal what
happens with the cellular immune response after the primary IVAG
infection with a wild type strain of the virus. Other studies involved
mice that were depleted in different T cell subpopulations by
inoculation with specific monoclonal antibodies to eliminate each one
of the T cell clones7. Although this method can elicit some
information about the role of T cells during HSV-2 infection,
interpretation is difficult because there is no guarantee that
depletion is complete. Instead, more reproducible results can be
obtained using congenitally athymic mice.
This report describes a straightforward study of the role of the T
cell immune response after wild type HSV-2-IVAG infection, using
genetically athymic (nude) mice, compared with their euthymic
counterpart. In order to study the progression of HSV-2 from the
vagina towards other organs in athymic mice, the presence of virus was
monitored by the peroxidase-antiperoxidase technique (PAP),
trans-mission electron microscopy and virus isolation.
Materials and methods
Virus. The ATCC VR-734 strain of Herpes simplex-2 was used. Virus
stock was prepared by inoculation of Vero cell monolayers grown in
plastic flasks, and maintained with Minimal Essential Medium (Gibco)
supplemented with 5% calf serum. When 80% of the cells showed
cytopathic effect (CPE), cultures were harvested, frozen and thawed 3
times, and spun down at 400 g. The supernatant was aliquoted, and
stored at -70°C until used. Virus stock was titrated using the plaque
forming unit method in Vero cell monolayers covered with culture
medium and methyl-cellulose.
Animals. Euthymic and athymic (nu/nu) N:NIH(S) (NIH) mice, and
euthymic and athymic (nu/nu) Balb/c females were obtained from the
bioterium of the Comisión Nacional de Energía Atómica, Argentina.
Mice were kept 5 to a box, and fed on pellets ad-libitum. Water, food
and cages were sterile, and the animals were maintained at constant
temperature, with natural cycles of light and darkness.
Experimental design. Every experiment included 20 animals of each
type. Ten week-old mice were gently swabbed IVAG with sterile, dry
cotton wool and inoculated at once with 5 x 105 pfu of HSV-2 suspended
in 0.05 ml of cell-culture medium. Clinical signs were recorded daily.
At two different timepoints [5 days post-inoculation (pi), and in the
premortem stage], animals were bled to death after ether anesthesia.
Heparinized blood samples were taken from each animal, and kept
separately at -70°C until detection of viremia by adsorption on Vero
cell monolayers and titration by pfu. A complete necropsy was
performed on each animal as follows: 5 consecutive transversal
sections of the whole body (each one 5 mm thick) were taken from vulva
to the lower kidney tip. The other organs were dissected separately,
and included liver, kidney, spleen, lung, heart, skin, brain, and bone
marrow. All the samples were immediately fixed in Bouin’s fluid for
6 h, then embedded in paraffin. Sections were stained with
Hematoxilin-Eosin, and serial adjacent sections were immunolabeled
with the PAP technique. This strategy of dissection allowed the
detection of HSV-2 antigens in every pelvic and abdominal organ
(including the spinal cord in situ) maintaining their normal
topographic location, which included the nerves and blood vessels.
Immunocytochemistry. The PAP method was perfor-med as previously
described12, using Dako polyclonal immunosera. The primary was a
rabbit immunoserum against HSV-2. Brains of intracerebrally infected
or normal uninfected mice embedded in paraffin were used as positive
and negative controls respectively.
Electron Microscopy. Suitable samples from different organs were taken
immediately after animal sacrifice, minced into 0.5 mm pieces and
fixed in 4% parafor-maldehyde-1% glutaraldehyde in PBS pH 7.4,
post-fixed in osmium tetroxide and embedded in Vestopal. Sections were
stained with uranyl acetate and lead citrate, and observed in a Zeiss
EM 109-T electron microscope with an acceleration of 80 KV.
Clinical signs and mortality in euthymic and athymic mice after
Fourteen out of 20 (70%) nude NIH mice, 6/20 (30%) euthymic NIH
mice, 16/20 (80%) nude Balb/c mice, and 16/20 (80%) euthymic Balb/c
mice developed signs of genital and neurological infection. All
animals that showed clinical signs of infection died (Fig. 1).
Spontaneous regression of infection after the appearance of disease
was not seen. At 4-6 days pi mice showed vulvar erythema, edema,
congestion and vaginal flux. At 6-7 days pi extensive ulcers were
observed in the vulvar mucosa and perivulvar and perianal area. In the
case of the euthymic mice, these ulcers also included alopecia. The
size and clinical appearance of the lesions were similar in euthymic
and athymic mice. At this same time, abdominal distension was also
observed in all the mice. At 7-8 days pi hind limb paresia appeared,
and death occurred by 8-10 days pi with a terminal picture that
included wheezing and lethargy.
HSV-2 progression and histologic lesions in
IVAG-infected euthymic and athymic mice
Viremia was not detected in athymic or euthymc mice of the NIH and
the Balb/c strains, whereas lesions and antigen distribution were
similar in the four experimental groups. HSV-2-PAP-positive areas were
observed by day 5 pi in the vulvar, vaginal and perianal skin
epithelia (Fig. 2A), in neurons belonging to autonomic ganglia located
near the vagina and the urinary bladder (Fig. 2B), and in the Auerbach’s
plexus of the large bowel (Fig. 2C). HSV-2 antigen was also detected
in small perivaginal and perivesical nerves. At the premortem stage
(8-10 days pi), HSV-2 antigens were still located in the structures
and organs described above, but also in the dorsal root ganglia, in
Auerbach’s plexus of the whole large bowel from rectum to cecum, and
in the spinal cord (Fig. 2D). The spinal cord was infected from the
lumbar up to the cervical area in the gray and the white matter of
lateral and dorsal columns and horns. No virus antigen was present in
the brain, but the pons and medulla oblongata were infected
HSV-2 antigens were not detected in the other organs (liver, lung,
heart, kidney, skin, spleen and bone marrow) of athymic or euthymic
The histologic lesions coincided with the PAP-positive areas, and were
mainly necrotic cells, with scattered intranuclear inclusion bodies.
In all the mice (athymic or euthymic) there were mild inflammatory
exudates underlying the necrotic epithelia. This exudate was mainly
composed of neutrophils.
Electron microscopy showed HSV particles in the nuclei of neurons
belonging to Auerbach’s plexus (Fig. 3A) and in epithelial cells
(Fig. 3B), thus confirming the presence of complete HSV-2 virions and
not only HSV-2 antigens in the infected organs.
Genetically athymic mice were used to study the pathogenesis of
wild-type HSV-2 IVAG infection. The results show a clear difference in
the percentage of infection and mortality between the athymic NIH mice
and the euthymic animals of the same strain. While 70% of athymic NIH
mice IVAG-inoculated with a wild type strain of HSV-2 developed a
detectable infection and died, only 30% of NIH euthymic mice showed
the same susceptibility. This difference is significant, after
analysis with Fisher’s exact test (p < 0.02). The athymic (nu/nu)
NIH mice used in this experiment are reported to lack T helper cell
activity, and are unable to generate cytotoxic T cells14. Moreover,
several histologic and functional criteria such as inability to reject
cells and xenografts, failure to mount a graft-vs-host response,
negligible response to T cell mitogens, etc, demonstrated that nude
mice are severely depleted of thymic derived lymphocytes14. Given the
fact that the only genetic difference between the NIH nude mice and
the euthymic mice of the same strain is the lack of T cell response,
it seems clear that the T cell-mediated immunity plays a protective
role in the IVAG infection produced by wild type HSV-2 in this mouse
strain. Other authors7 have also described T cell-defence against
vaginal HSV-2 challenge in mice after IVAG vaccination with attenuated
(thymidine kinase-deleted) HSV-2. This is in agreement with the data
shown in this experiment but, furthermore, the results reported herein
strongly suggest that T cell immune response can also limit wild-type
HSV-2 primary IVAG infection without any previous immunization.
The period between the virus inoculation and the first clinical signs
of vulvovaginal infection in mice that developed acute disease was 4-5
days. Thus, the NIH mice that showed no signs of infection and
survived HSV-2 IVAG inoculatin should have developed a protective T
cell response in less than 4-5 days. It could be argued that this time
is not enough for a mouse to activate T lymphocytes. However, specific
cytokine-secreting T cells were reported to be present in draining
genital lymph nodes of mice 4 days after IVAG infection with HSV-215.
According to the results presented herein the T cell-mediated
resistance to HSV-2-IVAG infection seems to depend upon the mouse
strain since the Balb/c mice, contrarily to what has been described
for the NIH mice, showed no difference in infection and mortality
between the athymic and the euthymic animals.
In this experimental model, HSV-2 progression was exclusively limited
to the nervous system, on the basis of HSV-2 antigen distribution and
histology. The strategy of embedding serial transversal slices of
whole mouse bodies in paraffin allowed a detailed observation of
organs, nerves, and blood vessels in situ, maintaining their normal
anatomic location. This method, associated with the use of the PAP
technique, and considered together with the chronologic development of
clinical signs at two timepoints had previously been used12, 16, and
makes it possible to infer the sequence of HSV-2 infection.
In both athymic and euthymic mice, HSV-2 replicated in the genital
epithelium, in Auerbach’s plexus, in neurons of sympathetic ganglia,
and in the spinal cord, but not in the brain. These results indicate a
wide infection of nerves and structures mainly belonging to the
autonomic nervous system, as previously reported in the IVAG infection
of euthymic Balb/c mice12. The infection of the autonomic nervous
system can explain the protracted large bowel paralysis with fecal
retention as well as the urinary bladder distention with urine
retention observed in all the necropsies. The consecutive metabolic
disturbances might explain the pathophysiology leading to death rather
than encephalitis, given the lack of brain or cerebellar infection.
Viremia was not detected in any animal (athymic or euthymic) either at
5 days pi or at the premortem stage. The complete absence of HSV-2
antigens in liver, lungs, kidneys, bone marrow, skin, heart, spleen,
and other organs also suggests that HSV-2 viremia did not occur. The
conclusion is that even though the T cell-mediated immune response can
limit HSV-2 IVAG infection to the genital tract in NIH mice, it has no
role in preventing HSV-2 dissemination through the blood. Otherwise,
in the athymic mice, HSV-2 should have infected and produced necrotic
lesions in several organs, (especially the liver), as previously
described in mice intraperitoneally inoculated17, 18. This was not
observed in any of the NIH or Balb/c nude mice used in this
experiment. Interestingly, other authors have also reported that in
nude mice, cell-mediated immune response was essential for eliminating
HSV from a site of inoculation other than IVAG, for example the ear
pinna, but in no case was viremia detected in T-cell-depleted
The employment of genetically athymic mice in this new experimental
model permitted a detailed study of HSV-2 progression after IVAG
infection, and suggests that viremia and HSV-2 spreading towards
organs other than the genital tract is not controlled by the T cell
Acknowledgements: This work was partially supported by the
University of Buenos Aires and is included in the research program of
C.A.E.H. (Argentine Chapter of the International Herpes Management
Forum), sponsored by Glaxo Wellcome Argentina.
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Fig. 1.– Percentage of mortality in athymic and euthymic mice after
Fig. 2.– HSV-2 antigen detected by PAP method in A: vaginal
epithelium, B: neurons of autonomic ganglia, C: Auerbach’s plexus of
the large bowel, and D: spinal cord. The dark spots are the positive
areas. A, B and C are mildly stained with Hematoxilin. A, B and C: X
150; D: X 50.
Fig. 3.– A: HSV-2 particles in a neuron of Auerbach’s plexus.
Amyelinic autonomic axons are indicated by arrows. B: HSV-2 virions in
a vaginal epithelial cell. X 40.000.