Download Herpes Simplex Virus Infection in Human Monocyte Cultures: Dose

381
J. gen. ViroL (1981), 52, 381-385
Printed in Great Britain
Herpes Simplex Virus Infection in Human Monocyte Cultures:
Dose-dependent Inhibition of Monocyte Differentiation Resulting in
Abortive Infection
(Accepted 22 September 1980)
SUMMARY
Monocyte-enriched cultures of human blood leukocytes were exposed to herpes
simplex virus (HSV) at different multiplicities of infection (m.o.i., from about 10 to
0.0001 p.f.u./cell). Highest maximum progeny virus titres were invariably obtained
with low initial m.o.i., i.e. those between 0.01 and 0.0001 p.f.u./cell, while little if any
infectious progeny was produced in cultures inoculated with the highest virus
concentrations. By the time of maximum virus production, i.e. 5 to 7 days after
inoculation, monocytes in the uninfected cultures had mostly differentiated to
macrophages. This differentiation was partially inhibited in cultures initially exposed
to the higher concentrations of HSV. Synthesis of HSV antigens was detected by
indirect immunofluorescence both in the high m.o.i, cultures and in the productively
infected cultures. By this criterion, a maximum of 10 to 15 % of all adherent cells
became infected in both culture types. It is suggested that the higher doses of HSV,
by inhibiting cellular maturation, also prevent the subsequent completion of its own
infectious cycle.
Mononuclear phagocytes have been shown to have a central role in the pathogenesis of
several experimental virus infections in animals. Genetic resistance of mice to certain virus
infections (Bang & Warwick, 1960; Goodman & Koprowski, 1962; Lindenmann et al.,
1978), as well as the post-natal maturation (Johnson, 1964; Zisman et al., 1970) of the in
vivo host defences towards experimental intraperitoneal virus infections, coincide with a
similar in vitro resistance or maturation of peritoneal macrophages from the respective inbred
strains of mouse (Mogensen, 1979). Spreading of an intraperitoneal herpes simplex virus
(HSV) infection in mouse to the liver (Mogensen, 1977) as well as to the central nervous
system (Johnson, 1964) is also determined by the susceptibility of peritoneal macrophages to
the infection. Much less information is available about the pathogenetic significance of
virus-mononuclear phagocyte interactions in the infections of man. It is known that in
generalized HSV infection the virus can harbour and spread throughout the body in the
leukocyte fraction of blood (Graig & Nahmias, 1973), but the actual host cell type involved in
this phenomenon has not been identified. According to previous reports, neither unstimulated
lymphocytes nor freshly isolated monocytes are able to support the growth of HSV in vitro.
After mitogenic stimulation, however, both T- and B-lymphocytes seem to be suitable hosts
for virus propagation (Kirchner et al., 1977; Kirchner & Schr6der, 1979). Human monocytes
incubated in culture for a few days transform to macrophages, and according to recent
reports, concomitantly acquire the capability to support HSV replication (Daniels et al.,
1978; Plaeger-Marshall & Smith, 1978).
In the present experiments cultures of human monocyte-enriched blood leukocytes were
prepared as described by Alitalo et al. (1980). More than 90% of the cells were initially
phagocytic (Hovi et al., 1977) and/or positive in the non-specific esterase (ANAE)
histochemical staining (Horwitz et al., 1977). The percentage of cells showing either or both
of these monocyte markers
further increased during culture.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 06 May 2017 23:12:05
0022-1317/81/0000-4286 $02.00 © 1981 SGM
Short communications
382
5
i
~
u
i
[
i
Q
,
0
1
'\J
",.
2
3
4
5
6
Time after infection (days)
Fig. 1. Replication of HSV- 1 in human monocyte cultures. One-day-old monocyte-enriched leukocyte
cultures were infected with different dilutions of HSV-1 produced in human embryonal skin fibroblasts,
titre 2-6 x 106 p.f.u./ml. Twenty-five ltl of 10-fold dilutions of virus were inoculated in Linbro wells
(1.5 ml of growth medium). Approximate initial multiplicities of infection (p.f.u./cell): •
• , 0.1;
m - - - I , 0.01; O - - . - - O , 0.001; IS3~--IS], 0.0001. Titres indicated represent total infectious virus.
Arrow at symbol means a titre as high as or higher than indicated. The quantification of infectious virus
was done by a standard plaque assay technique (Vaheri et al., 1969) using cultures of BSC-1 cells• Cell
monolayers on Petri dishes were inoculated with 0-2 ml portions of 10-fotd dilutions of the preparation
to be tested. Plaques were counted after 4 to 5 days of incubation.
Fresh cultures (~<24 h) were inoculated with HSV type 1 (HSV-1) preparations at different
multiplicities of infection (between about 10 and 0.0001 p.f.u./cell, 10-fold dilutions of virus
in each experiment) and incubated at 37 °C. The stock virus preparations were of H S V - I ,
Tyler strain, propagated with diluted inocula (m.o.i. about 0.1 p.f.u./cell) in cultures of
human embryonal skin fibroblasts. The cultures inoculated with the highest multiplicities
(from 10 to 0.1 p.f.u./cell) produced little, if any, infectious progeny virus, while diluted
inocula (from about 0.01 to 0.0001 p.f.u./cell) readily replicated in the cultures (Fig. 1). The
maximum progeny virus titres were obtained about 5 to 7 days after inoculation.
The monocyte origin of the virus-producing cells was documented by first staining for HSV
antigen by indirect immunofluorescence (Leinikki, 1973) and then for A N A E . Cells showing
HSV-specific fluorescence were constantly positive for the A N A E marker.
It could be argued that in the case of productive infection the virus initially did not adsorb
to monocytes but remained in the culture medium until sufficient differentiation had taken
place. This is unlikely, however, because practically no infectious virus could be detected in
these cultures during the first few days of infection. Furthermore, some cells (~<1%)
containing HSV antigens, as judged by indirect immunofluorescence, were seen in the low
m.o.i, cultures even after 24 h of infection, indicating that the infectious cycle was initiated
while still at the monocyte stage. The percentage of cells containing virus antigen increased
with time, reaching a maximum of 10 to 15 % by the end of the observation period. In the
high m.o.i, cultures the maximum percentage of fluorescent cells (10 to 15 %) was seen within
the first 1 to 2 days of infection. During the subsequent 4 to 5 days, the proportion of cells
containing HSV antigen decreased below 5 %.
It is known that defective interfering (DI) particles can be generated during HSV infection
in vitro, especially by successive undiluted passages (e.g. Schr6der & Urbaczka, 1978). Our
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 06 May 2017 23:12:05
Short communications
383
HSV-1 laboratory strain had been kept in BSC-1 or human skin fibroblasts for a number of
passages without any signs of the presence of DI particles. To investigate this possibility in
the present virus preparations, 10-fold dilutions of virus, resulting in multiplicities similar to
those used in monocyte cultures, were inoculated into cultures of human skin fibroblasts. No
high multiplicity interference was seen in these fibroblast cultures. Furthermore, the
phenomenon in monocytes was equally demonstrated with a fresh HSV-1 isolate kindly
provided by the Diagnostic Laboratory of this Department, propagated for only two passages
in cultures of human embryonic skin fibroblasts. These results suggest that DI particles do
not play a major role in generation of the high multiplicity interference of HSV replication in
monocyte cultures. However, they do not exclude the possibility that DI particles,
presumably present in small amounts in the inoculum virus are, for some reason, replicated
particularly efficiently in monocytes or alternatively, that replication of HSV is more sensitive
to the interfering action of DI particles in monocytes than e.g. in fibroblasts.
The high multiplicity interference could have been due to interferon or some other putative
inhibitory soluble contaminant (Morahan et al., 1980) in the crude inoculum virus
preparations. To test this possibility we produced partially purified virus preparations by first
centrifuging infected culture media at 10000 g for 10 min and then pelleting the virus from
the supernatant by centrifugation at 22000 rev/min in a Beckman SW27 rotor for 75 min. A
similar high multiplicity interference was obtained with these virus preparations as in cultures
infected with the crude stock virus. Thus a putative soluble contaminant in the inoculum does
not seem to cause this phenomenon. Neither was the phenomenon dependent on the host cell
type used to propagate the inoculum virus. High multiplicity interference was constantly
observed with all tested stock virus preparations, i.e. HSV-1 preparations produced in adult
or embryonic human skin fibroblasts, BS-C-1, Vero or L-132 cells or in monocyte cultures, as
well as with the prototype strain of HSV type 2 obtained from the American Type Culture
Collection and propagated in our laboratory for two passages in human skin fibroblasts.
Another alternative for the high multiplicity interference could be the inhibition of cellular
differentiation observed in the high-dose HSV-infected monocyte cultures. During the
experiments, cultures were inspected daily with a light microscope and the morphological
differentiation to macrophage-like cells was recorded. The majority of the cells in the
uninfected cultures was found to differentiate to macrophages within a 1 week incubation
period (Alitalo et al., 1980). HSV-1 infection at higher multiplicities (from 10 to 0.1) inhibited
this differentiation in a dose-dependent way, with most of the cells exposed to the highest
virus doses remaining small and round (Fig. 2). These cultures also showed a greater
tendency for cell detachment than the uninfected ones. In contrast, cultures showing the
optimum virus replication were indistinguishable in microscopic morphology from the
controls.
It has been reported that human monocytes cultured in vitro for a few days acquire the
capability to support HSV replication (Daniels et al., 1978; Plaeger-Marshall & Smith, 1978).
In our studies infectious progeny virus was recovered from the productively infected cultures
only after 2 to 3 days incubation (Fig. 1) when at least some of the cells already showed
evident signs of differentiation.
The presence of virus antigens in the high m.o.i, cultures together with lack of infectious
virus progeny can be taken as evidence for an abortive infection, in accordance with the
earlier report of Daniels et al. (1978).
These results suggest that exposure of human monocytes to a relatively high dose of HSV
results in inhibition of cellular maturation which in turn prevents completion of the infectious
cycle of the virus, resulting in abortive infection.
The mechanism of HSV-induced inhibition of monocyte differentiation is not known but
one plausible candidateDownloaded
is interferon,
induced by HSV
frompresumably
www.microbiologyresearch.org
by infection in monocytes.
IP: 88.99.165.207
On: Sat, 06 May 2017 23:12:05
Short communications
384
'+ . - -
~
.~, ~
..-
++~,+" + "++
" ~'-+~_'11
~
6 0
+-"
+
er~' e
+ ,+,~+. ..+ : .0
s ,maP..P'+_,~,,+,, ~
,,+',+
I~_L~
+
+
•
+
.
'+m
,,,.
+
t r,.+
,+, +..+p. ,.I
+¢.,+
_ v
"~
"31s~+ ..,. ~ + _ + , , . "+e,~ +~
~
"wF
+ +~'.~++" ++, ++. %+.+. +++, ,,,+
"
~+?+"
++ . . L ~
+ P
|
+ +++.,+~,.+++"" + "r. ,+...
• ,,,.~l~+m ~,,,' e. l,+,+,..+%++ r~
O
....
+I,
.
+ ,. + +
+
+
+
<+,> ".~,~+.+
.. :+~ i. , ~
+
m~F~'~.~
+
+
+
,F
.
II!,IF
"
~
'+
,++
+
-
+-+~.;
,~+
B+..+ ~++.
+
++++
++
+.+
,+ ~"+ I
~"
. + ~ .
.
+
~r ~
+',~
Fig, 2. Inhibition of monocyte differentiation by high multiplicity HSV infection. Monocyte cultures (see
text and Fig. 1) were infected with different m.o.i, of HSV-1, Photographs were taken after 6 days
incubation and May-Griinwald-Giemsa staining, (a) A high m.o,i, culture without infectious virus
progeny. Cells are small like fresh monocytes. (b) A productively infected culture inoculated with a
multiplicity 10-4 of that in (a). Cells have differentiated to macrophages+ (c) Control culture without
virus infection and showing
typical macrophages,
Downloaded
from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 06 May 2017 23:12:05
Short eolnmunications
385
This view is supported by the fact that relatively low doses of purified human leukocyte
interferon added to monocyte cultures can reversibly induce an inhibition of monocyte
maturation morphologically similar to that described in the present paper (Lee & Epstein,
1980; T. Hovi et al., unpublished results). Studies on the possible induction of interferon by
HSV infection in monocyte cultures are in progress.
This work was supported by the Medical Research Council of the Academy of Finland and by the
Finnish Cancer Foundation. We are grateful to Miss Helena Kainulainen for technical assistance and
to Dr O. Saksela for preparing the photographs.
Department of Virology, University of
Helsinki, Haartmaninkatu 3
SF-00290 Helsinki 29, Finland
K I M M O LINNAVUORI
T A P A N I HOVI*
REFERENCES
ALITALO, K., HOVI, T. & VAHERI, A. (1980). Fibronectin is produced by h u m a n macrophages in culture. Journal of
Experimental Medicine 15 l, 602-613.
BANG, G. B. & WARWICK,A. (1960). Mouse macrophages as host cells for the mouse hepatitis virus and the genetic
basis of their susceptibility. Proceedings of the National Academy of Sciences of the United States of America
48, 1065-1975.
DANIELS, C. A., KLE1NERMAN, E. S. & SNYDERMAN, R. (1978). Abortive and productive infectious of human
mononuclear phagocytes by type 1 herpes simplex virus. A merican Journal of Pathology 9, 119-129.
GOODMAN, G. T, & KOPROWSKI, n. (1962). Macrophages as a cellular expression of inherited natural resistance.
Proceedings of the National Academy of Sciences of the United States of America 48, 160-165.
GRAIG, C. P. & NAHMIAS, A. J. (1973). Different patterns of neurologic involvement with herpes simplex virus types
1 and 2: isolation of herpes simplex virus type 2 from the buffy coat of two adults with meningitis. Journal of
Infectious Diseases 127, 365-372.
HORWITZ, D. A., ALLISON, A. C., WARD, P. & KNIGHT, N. (1977). Identification of h u m a n mononuclear leukocyte
populations by esterase staining. Clinical and Experimental Immunology 30, 289-298.
HOV1, T,, MOSHER, D. & VAHERI, A. (1977). Cultured h u m a n monocytes synthesize and secrete a2-macroglobulin.
Journal of Experimental Medicine 145, 1580-1589.
JOHNSON, R. T. (1964). The pathogenesis of herpes virus encephalitis. II. A cellular basis for the development of
resistance with age. Journal of Experimental Medicine 120, 359-374.
KIRCHNER, H. & SCHR()DER, C. H. (1979). Replication of herpes simplex virus in h u m a n B lymphocytes stimulated
by Epstein Barr virus. Intervirology 11, 61-65.
K1RCHNER, H., KLEINICKE, C. & NORTHOFF, H. (1977). Replication of herpes simplex virus in h u m a n peripheral T
lymphocytes. Journal of General Virology 37, 647-649.
LEE, S. I-1. S. & EPSTEIN, L. B. (1980). Reversible inhibition by interferon of the maturation of h u m a n peripheral
blood monocytes to macrophages. Cellular bnmunology 50, 177-190.
LEINIKKI, P. (1973). Typing of Herpesvirus hominis strains by indirect immunofluorescence and biological
markers. Acta Pathologica et Microbiologica Scandinavica 81, 65-69.
LINDENMANN, J., DEVEL, E., FUNCONI, S. & HALLER, O. (1978). Inborn resistance of mice to myxoviruses:
macrophages express phenotype in vitro. Journal of Experimental Medicine 147, 531-540.
MOGENSEN, S. C. (1977). Role of macrophages in hepatitis induced by herpes simplex virus types 1 and 2 in mice.
Infection and Immunity 15, 686-691.
MOGENSEN, S. C. (1979). Role of macrophages in natural resistance to virus infections. Microbiological Reviews
43, 1-26.
MORAHAN, P. S., MORSE, S. S. & MCGEORGE, M. B, (1980). Macrophage extrinsic antiviral activity during herpes
simplex virus infection. Journal of General Virology 46, 291-300.
OLAEGER-MARSHALL, S. & SMITH, J, W. (1978). Experimental infection of subpopulations of h u m a n peripheral
blood leukocytes by herpes simplex virus (40185). Proceedings of the Society for Experimental Biology and
Medicine 158, 263-268.
SCHR6DER, C. H. & URBACZKA,G. (1978). Excess of interfering particles over infectious particles in herpes simplex
virus passaged at high m,o.i, and their effect on single-cell survival. Journal of General Virology 4 1 , 4 9 3 - 5 0 1 .
VAHERI, A., VON BONSDORFF, C-H., VESIKARI, T., HOVI, T. & V.~.~N,~NEN, P. (1969). Purification of rubella virus
particles. Journal of General Virology 5, 39-46.
ZlSMAN, B., HIRSCH, M. C- & ALLISON, A. C. (1970). Selective effects of anti-macrophage serum, silica and
anti-lymphocyte serum on pathogenesis of herpes virus infection in young adult mice. Journal oflmmunology
104, 1155-1159.
4 June 1980)
Downloaded (Received
from www.microbiologyresearch.org
by
IP: 88.99.165.207
On: Sat, 06 May 2017 23:12:05