VIROLOGY 219, 357–366 (1996)
ARTICLE NO. 0261
Importance of Localized Skin Infection in Tick-Borne Encephalitis Virus Transmission
MILAN LABUDA,*JONATHAN M. AUSTYN,† EVA ZUFFOVA,*OTO KOZUCH,‡ NORBERT FUCHSBERGER,‡
JAN LYSY,*and PATRICIA A. NUTTALL§,1
*Institute of Zoology and ‡Institute of Virology, Slovak Academy of Sciences, 842 46 Bratislava, Slovakia; †Nuffield Department
of Surgery, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom; and §NERC Institute
of Virology and Environmental Microbiology, Oxford OX1 3SR, United Kingdom
Received December 28, 1995; accepted March 5, 1996
Arboviruses are transmitted to vertebrates by the ’’bite‘‘ of infected arthropods. Events at the site of virus deposition are
largely unknown despite increasing evidence that blood-sucking arthropods immunomodulate their skin site of feeding. This
question is particularly relevant for ixodid ticks that feed for several days. To examine events under conditions mimicking
tick-borne encephalitis (TBE) virus transmission in nature (i.e., infected and uninfected Ixodes ricinus ticks feeding on the
same animal), infected adult and uninfected nymphal ticks were placedin one retaining chamber (skin site A) and uninfected
nymphs were placed within a second chamber posteriorly (skin site B) on two natural host species, yellow-necked field
mice (Apodemus flavicollis)and bank voles (Clethrionomys glareolus). Virus transmission frominfected touninfected cofeed-
ing ticks was correlated with infection in the skin site of tick feeding. Furthermore, virus was recruited preferentially to the
site in whichticks were feeding compared with uninfested skin sites.Viremia did not correspond with a generalized infection
of the skin; virus was not detected in an uninfested skin site (C) of 12/13 natural hosts that had viremia levels §2.0 log10
ic mouse LD50/0.02 ml blood. To characterize infected cells, laboratory mouse strains were infested with infected ticks and
then explants were removed from selected skin sites and floated on culture medium. Numerous leukocytes were found to
migrate from the skin explants of tick feeding sites. Two-color immunocytochemistry revealed viral antigeninboth migratory
Langerhans cells and neutrophils; in addition, the migratory monocyte/macrophages were shown to produce infectious
virus. The results indicate that the local skin site of tick feeding is an important focus of viral replication early after TBE
virus transmission by ticks. Cellular infiltration of tick feeding sites, and the migration of cells from such sites, may provide
a vehicle for transmission between infected and uninfected cofeeding ticks that is independent of a patent viremia. The
data support the hypothesis that viremia is a product, rather than a prerequisite, of tick-borne virus transmission.
Academic Press, Inc.
INTRODUCTIONticks (Gresikova ´and Calisher, 1988). When infected and
uninfected vectors were allowed to feed together on
these hosts (mimicking natural conditions of virus trans-
mission), the greatest numbers of infected ticks were
obtained fromsusceptible host species (Apodemus spp.)
thathad low or undetectable levels of viremia (so-called
‘‘nonviremic transmission’’). In contrast, fewer infected
ticks were obtained by feeding on voles (Clethrionomys
and Pitymys spp.) which produced significant levels of
viremia (Labuda et al., 1993b). Nonviremic transmission
has been reproduced experimentally by inoculating tick-
infested animals with virus mixed with either salivary
glandextractorsaliva fromuninfectedfeeding ticks.This
phenomenon, named ‘‘saliva-activated transmission’’
(SAT), appeared to result from localized changes in the
skin induced by tick saliva (presumablyto facilitate feed-
ing). Such changes were somehow exploited by certain
viruses to promote transmission (reviewed by Nuttall et
Innatural infections with arthropod-borne flaviviruses,
or following experimental intradermal or subcutaneous
inoculation, virus first replicates at the inoculation site
and subsequently in lymph nodes that drain the site (Al-
Tick-borne encephalitis (TBE)virus (familyFlaviviridae,
genus Flavivirus), an important human pathogen, is en-
demic over a wide area covering parts of Europe, north-
ern Asia, and China. Ixodes ricinus is the primary tick
vector of the European subtype of TBE virus (reviewed
by Nuttall and Labuda, 1995). According to the definition
ofan arbovirus, tick vectors become infected when they
feed on the blood of a viremic host (WHO, 1986). Cur-
rently, the presence of a readily detectable viremia,
above a threshhold level to infect feeding vectors, is the
main criterion used to identify the amplifying hosts of
TBE virus in nature (Labuda et al., 1993b). However, ex-
perimentalstudies have demonstratedefficientTBE virus
transmission frominfected to uninfected ticks cofeeding
on vertebrate hosts that have undetectable or very low
levels ofviremia (Labudaetal., 1993a).InCentral Europe,
small rodents such as yellow-necked field mice (Apode-
mus flavicollis) andbank voles (Clethrionomys glareolus)
are commonly infested withimmature stages ofI. ricinus
1To whom reprint requests should be addressed.
Copyright? 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.
358LABUDA ET AL.
brecht, 1968). The specific cell types involved in viral
replication at the inoculation site, and the temporal se-
quence ofevents in the skin afterinfection, are unknown
(Monath, 1986). Since TBE virus is transmitted through
the skin, one possibility is that free virus passes to the
draining lymph nodes to establish the infection. Alterna-
tively, or in addition, the skin may be a primary site of
viral replication in the early phase of infection. In this
case, various cell types could potentially be infected by
TBE virus,including epidermal Langerhans cells (LC)and
keratinocytes,and dermal macrophages and neutrophils
that are recruited during the inflammatory response to
thetick bite (Wikeletal.,1994).LC ofskinare members of
the dendritic cell (DC) system(Austyn, 1992). Inprevious
studies of skin explants in culture, LC were observed to
migrate fromthe epidermis, through the dermis, and out
of the tissue, to accumulate at the bottom of the culture
vessel (Larsen et al., 1990); in vivo, these cells enterthe
draining lymph nodes (M. I. Liddington and J. M. Austyn,
unpublished data). In principle, such migratory cells
could also be responsible for transporting virus into
lymph nodes of the host.
In order to better understand the role ofskin infection
in TBE virus transmission and the mechanismof SAT, a
comparative study ofvirus dissemination in the skin and
virus transmission between cofeeding ticks was per-
formed. Virus transmission efficiency was estimated by
the percentage of I. ricinus nymphs acquiring infection
during different intervals of cofeeding. The skin infection
was investigated using several different approaches: di-
rect assay ofhomogenized skin samples, culture of skin
explants followedbyvirus titration ofthe culture medium,
andscreening ofcells emigrating fromskin explants with
an emphasis on LC. In addition, the ability of TBE virus
to infect LC in vitro was determined.
serum (FBS), 2 mM glutamine, 45 mg/ml penicillin, 45
mg/ml streptomycin, and 90 mg/ml kanamycin (complete
RPMI medium). Pig stable kidney (PS) cells were propa-
gated in L-15 medium supplemented with 5% FBS and
antibiotics. During virus assays, PS cells were cultured
in Earle’s modification of Eagle’s medium (EMEM) sup-
plemented with 3%FBS for 4 days at 37?.
Yellow-neckedfield mice andbank voles were trapped
in areas of Slovakia considered free of TBE virus and
tested for neutralizing antibodies to TBE virus prior to
use in the experiments. No animal was shown to have
antibodies to TBE virus.
For further study of TBE virus infection of host skin
withthe emphasis on cell types involved inthe infection,
laboratory mice of two strains were used. Male C57Bl/6
(H-2b) mice were obtained fromHarlan UK (Bicester, UK)
and Balb/c (H-2d) mice were purchased fromthe Sir Wil-
UK). The animals were more than 8 weeks old when
used. The use ofthese mouse strains allowed us to use
a monoclonal antibody to identify LC.
Infection and transmission experiments
For the cofeeding experiments, yellow-necked mice,
bank voles, and laboratory mice were each infested with
two pairs of adult I. ricinus ticks (virus-infected females
and uninfected males) held within a retaining chamber
attached to the dorsal surface of trunk skin (chamberA).
In the same chamber as the adult ticks, 20 uninfected
nymphs were added. An additional 20 uninfected I. ri-
cinus nymphs were placedina separate retaining cham-
ber(chamberB) onthe dorsal surface posterior to cham-
ber A. The minimum distance between ticks feeding in
chambers A and B was approximately 1 cm. Ticks were
allowedtofeed for1,2,or3 days andwere thenremoved
by traction from humanely killed animals, weighed, and
stored. To examine the effect of delaying nymphal feed-
ing, nymphs were added to chamber B 1 day after in-
fected adults and uninfected nymphs were added to
chamberA; onthe following day,all ticks were collected.
Thus nymphs in chamber A fed for 2 days, coinciding
with the 2 days in which infected adults fed, whereas
nymphs in chamber B fed for only 1 day, coinciding with
the 2nd day ofadult feeding. Forcomparison, some ani-
mals had no ticks placed in chamber B. Blood samples
(0.1 ml of blood in 0.9 ml of complete EMEM for plaque
titration and 0.2 ml of blood in 0.2 ml of 1% heparin for
intracranial mouse inoculation) were obtained by heart
puncture or puncture of the sinus orbitalis from each
experimental animal (no difference in virus titers was
detected for the two blood sources).
Field mouse and bank vole skin samples of4 1 5 mm
(20 mm2) and approximately 15 mg weight were excised
MATERIALS AND METHODS
I. ricinusnymphs andadults were collectedbyflagging
the vegetation in areas (southern England and selected
sites of southwestern Slovakia) where TBE virus has
never been detected. First-generation ticks fed in the
laboratoryonoutbredguinea pigs andrabbits wereused.
All the female I. ricinus used in the experiments were
infected with a European subtype of TBE virus (isolate
198 obtained from I. ricinus ticks collected in Slovakia
and passaged 26 times in suckling-mouse brain) by par-
enteral inoculation (mean titer 3.0 log10plaque-forming
units (PFU)/tick ofTBE virus suckling-mousebrainstock).
The inoculated ticks were incubated for 14 days prior to
use in the experiments.
Cells derived from rodent tissues were cultured in
RPMI1640 mediumsupplemented with 10%fetal bovine
366 LABUDA ET AL.
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these cells, togetherwithLC migrating fromthe explants,
were ultimatelyinfected withTBE virus, and both epider-
mal LC and keratinocytes could be infected in vitro (Fig.
1). Thus the inflammatory response perse may increase
the probability that TBE virus meets cells susceptible to
infection. This effect, combined with local immunomodu-
lationinducedbytick saliva,couldfacilitatethe establish-
ment of an infection at the site of infected tick feeding.
Suchimmunomodulatory effects thatare potentially ben-
eficial to the virus include suppression of natural killer
cell activity (Kubes ˇ et al., 1994) and cytokine activity/
production (Wikel et al., 1994; Fuchsberger et al., 1995).
Similarly, cellular infiltration in response to inflammation
could benefit the virus by recruiting infected cells to the
skin site of uninfected tick feeding. This would explain
the preferential attraction ofvirus to the skin sites oftick
feeding. Whether or not the neutrophils and monocytes
that are recruited at later time points are infected prior
to their entry to the skin awaits further investigation.
Detection ofTBE viral antigenin LC suggests a poten-
tial vehicle for virus dissemination. Epidermal DC (i.e.,
LC) internalize and process locally acquiredforeignanti-
gens, express the relevant peptide–MHC complexes,
and migrate to draining lymph nodes where they deliver
costimulatory signals for T cell activation and the initia-
tion of primary immune responses (Larsen et al., 1990;
Steinman, 1991; Austyn, 1992). Although the results do
not prove that TBE virus established a productive infec-
tion in LC, the level of staining of viral antigen strongly
suggests active viral replication in the cells rather than
noninfectiveprocessing ofvirus bythese cells.Anintrigu-
ing possibility is thatcomponents oftick saliva modulate
LC function, perhaps to decrease the host’s immune re-
sponse against the tick. Such an immunomodulatory ef-
fectmight be exploited by the virus to facilitate infection
of LC, increasing the cytopathogenicity of TBE virus for
LC as suggested by the observed syncytial formation in
vivo but not in vitro.
We thank Dr.E.A.Gould(NERC InstituteofVirologyandEnvironmen-
tal Microbiology, Oxford) for providing hyperimmune anti-TBE rabbit
serum, and Mrs. D. Hankins and Mr. M. I. Liddington for technical
assistance. The work was supported by a European Commission Hu-
man Capital and Mobility Programme Fellowship awarded to Dr. La-
buda, by NATO Linkage Grant 940225, and by Slovak Grant Agency
for Science Grant 1244/95.
Albrecht, P. (1968). Pathogenesis of neurotropic arbovirus infections.
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