Download Isolation of Haemorrhagic Fever with Renal Syndrome Virus from

J. gen. Virol. (1985), 66, 1271-1278. Printed in Great Britain
1271
Key words: HFRS/Hantaan virus~rats
Isolation of Haemorrhagic Fever with Renal Syndrome Virus from
Leukocytes of Rats and Virus Replication in Cultures of Rat and Human
Macrophages
ByTAKAO
NAGAI, I OSAMU TANISHITA, 2 YOSHIYUKI TAKAHASHI, 2
T A K A H I S A Y A M A N O U C H I , 3,~ K A Y O K O D O M A E , 4
KAZUHIROKONDO,
s J O S E R. D A N T A S , JR, 5 M I C H I A K I T A K A H A S H I 5
AND K O I C H I Y A M A N I S H I s*
1Department of Pediatrics, Fujita-gakuen Hoken-eisei University, School of Medicine, Toyoake,
Aichi 470-11, ZKannonji Institute, Research Foundation for Microbial Diseases, Osaka
University, Kannonji, Kagawa 768, 3Department of Tuberculosis Research 11, 4Quarters for
Experimentally Infected Animals and SDepartment of Virology, Research Institute for Microbial
Diseases, Osaka University, Suita, Osaka 565, Japan
(Accepted 8 February 1985)
SUMMARY
Newborn rats were inoculated intraperitoneally with haemorrhagic fever with renal
syndrome (HFRS)-related virus (B-1 strain), and virus isolation from their various
organs was attempted between 1 and 25 weeks after inoculation. Virus could be isolated
repeatedly from lung, brain, spleen and kidney and also from peripheral blood. When
virus isolation was carried out on fractionated peripheral blood cells, virus was
associated mainly with the macrophage fraction and to a lesser extent with
granulocytes. Virus replicated well in peritoneal exudate cells of normal rats and it grew
in the adherent mononuclear cells from normal human peripheral blood. These data
suggest that macrophages, permissive for HFRS-related virus replication, might
contribute to the spread of viral infection in vivo.
INTRODUcTIoN
Haemorrhagic fever with renal syndrome (HFRS) has been reported in Scandinavia
(Myhrman, 1951 ; Laedvirta, 1971), the Soviet Union (Casals et al., 1966) and the north of China
(Ishii et al., 1942), and has been well known since 1951 as Korean haemorrhagic fever because of
epidemics among United Nations troops in Korea (Smadel, 1953). Recently, there have been
several reports of similar diseases in other areas (Cohen et al., 1981 ; Desmyter et al., 1983 ; Mery
et al., 1983).
A viral agent, Hantaan virus (HV), was first isolated from the lungs of Apodemus agrarius
coreae, a rodent reservoir, and it was proved that HV was the aetiological agent of Korean
haemorrhagic fever by serological tests by Lee et al. (1978). Thereafter, HFRS-related viruses,
which were antigenically related to HV, were isolated in various countries from wild and
laboratory rodents (Lee et al., 1982a, b; Kitamura et al., 1983; Song et al., 1983; Niklasson &
LeDuc, 1984; Yanagihara et al., 1984). In Japan more than 120 cases of this disease were
reported between 1970 and 1984 from many medical institutions (Kawamata, 1983). We have
made an independent isolation of a Hantaan-related virus (B-1 strain) from a tumour removed
from a laboratory rat kept by a patient who developed clinical HFRS (Yamanishi et al., 1983).
As a result of virological and pathological studies of animals infected with this virus, it was
confirmed that systemic infection occurs in rats (Kurata et al., unpublished data). Recently,
one of our colleagues, who had been involved in animal experiments, was accidentally bitten
by a rat infected with the B-1 strain and typical HFRS developed in 2 weeks. Since the
antibody titre in his serum increased, it is apparent that the B-1 strain can cause HFRS in man
(unpublished data). Virus could be isolated from the blood of the patient, although it is presently
0000-6475 © 1985 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 18:10:33
1272
T. N A G A I AND OTHERS
unclear in which leukocyte fraction HFRS virus resides. Interaction of HFRS-related virus with
leukocytes may be important in relation to systemic infection and also in the pathogenesis of this
disease. In this report, virus isolation from various rat organs including fractionated leukocytes
is described and, in addition, virus replication in mononuclear cells from normal human
peripheral blood and in peritoneal exudate cells from rats was investigated.
METHODS
Cells. Vero E6 cells were obtained from the American Type Culture Collection and cultured in growth medium
containing a mixture of Medium 199 and Eagle's MEM supplemented with 50/0 foetal calf serum (FCS) or in
maintenance medium which had the same composition but with 3 ~ FCS.
Virus and eirus titration. The B°I strain was used. This strain was originally isolated in cell culture from a tumour
specimen of a rat kept in a medical institution (Yamanishi et al., 1983), passaged 6 times in Vero E6 cells and
prepared as stock virus. The virus titre was measured by a direct immunofluorescence (IF) method as follows. Vero
E6 cells in Lab-Tek 8 chambers (Miles Laboratories) were inoculated with diluted virus solution and adsorbed for
1 h at 37 °C. The cultures were overlaid with the maintenance medium containing 1.3 ~ methyl cellulose, and then
kept for 7 days at 37 °C in a CO_, incubator and fixed in a mixture of 90 ~ acetone and 10 ~ methanol for 15 min at
4 °C. IF tests were performed using clone 80 monoclonal antibody. The isolation and characterization of this
monoclonal antibody has been reported previously (Yamanishi et al., 1984). Briefly, BALB/c mice were
immunized with lysates of Hantaan virus (76-118 strain)-infected cells. Hybridomas were obtained from the
cultures of SP/2 myeloma cells fused with spleen cells from immunized mice. Twenty clones were obtained and
clone 80 reacted with a polypeptide of tool. wt. approx. 50000, presumably corresponding to the viral nucleocapsid
protein. Antibody from ascites was purified by centrifugation followed by precipitation with ammonium sulphate,
and labelled with ftuorescein isothiocyanate (FITC). The number of foci was counted under the fluorescence
microscope and the titre was expressed as the number of focus-forming units (f.f.u.) per ml.
Animals. Pregnant Fisher rats (F344/N strain), which were obtained from Shizuoka Laboratory Animal Center
and kept in our Institute, were used. Three litters of newborn rats (total of 24 rats per experiment) were inoculated
intraperitoneally (i.p.) with 0.05 ml of virus (8.5 × 102 f.f.u./rat).
Virus isolation. Virus isolation from brain, lung, spleen, kidney and peripheral blood of newborn rats was
attempted at 1,3, 5, 8, 11, 15, 20 and 25 weeks after inoculation. Each organ was not homogenized but minced, and
put on to Vero E6 cells in plastic dishes. After 1 day of cultivation, cultures were washed to remove minced pieces
of organs and then kept at 37 °C in a CO2 incubator. Cells were trypsinized at 1 or 2 weeks after inoculation and
seeded onto 12-well spot-slides. Subsequently, after overnight incubation, the ceils were fixed with a mixture of
90°0 acetone and 10~ methanol for 15 min at 4 °C and direct IF tests were performed as described above.
Fraetionation oJ'rat or human peripheral leukocytes. Human peripheral blood was obtained from healthy donors
who h a d n o evidence of HFRS and had no antibodies in their sera against the B-I strain virus. Rat peripheral
blood was collected by heart puncture at 5 weeks post-infection. Rat and human peripheral blood were collected in
heparinized syringes and separated as described elsewhere (Kumagai et al., 1979).
Briefly, peripheral mononuclear cells were obtained by centrifugation on Lymphocyte Separation Medium
(Litton Bionetics Laboratory Products, Kensington, Md., U.S.A.). After washing twice in RPMI 1640 medium by
low-speed centrifugation, the cells were suspended in RPMI 1640 medium supplemented with 10°~ FCS. This
suspension was placed in plastic dishes which had been coated with FCS by overnight incubation at 4 °C, then
incubated for 2 h at 37 °C. Non-adherent cells were collected, washed and suspended in RPMI 1640 medium
supplemented with 10~o FCS adjusting the concentration of cells to about 1 x 106 cells/ml. Non-adherent cells
were used as the lymphocyte fraction. Adherent cells were detached from the plastic plates by incubation at 4 °C in
phosphate-buffered saline (PBS) containing 0.2~ EDTA and 5% FCS, and then, after washing twice in RPMI
1640 medium, suspended in RPMI 1640 medium supplemented with 10% FCS so as to adjust the concentration of
cells to about 1 x l0 s cells/ml. Adherent cells were used as the macrophage fraction.
For activation of lymphocytes, non-adherent cells were cultured with medium containing 0.015 mg/ml
phytohaemagglutinin P (PHA-P) (Difco) for 3 days before virus inoculation, and the cells were cultured with
RPMI 1640 medium containing 10~ FCS and 20~o Lymphocult-T (Biotest Diagnostics, F.R.G.) after
inoculation. For activation of macrophages, adherent cells were cultured for 3 days with 0.001 mg/ml
lipopolysaccharide (LPS) (Difco), which has an ability to stimulate macrophage function (Allison et al., 1973;
Hotta & Hotta, 1982), and infected with virus.
Rat granulocytes from peripheral blood were obtained by additional centrifugation on 7 5 ~ Percoll
(Pharmacia). The purity of the granulocytes was assessed by Giemsa staining and was more than 95%
Peritonealexudate cells (PEC). Healthy Fisher rats aged 6 weeks were used for the collection of PEC. At 3 days
after i.p. injection of thioglycollate, rats were sacrificed by heart puncture and PEC were obtained. The cells were
suspended in RPMI 1640 medium supplemented with 10°o FCS and cultured in plastic dishes which had been
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 18:10:33
H F R S virus in rats
1273
coated with rat plasma for 3 days. Non-adherent cells were discarded and adherent cells were used as PEC. The
purity of the macrophages in PEC was tested as described elsewhere (Kumagai et al., 1979)and approached 100~.
Virus injection to mononuclear cellsJrom human peripheral blood and PEC from rats. Lymphocyte and macrophage
fractions of human peripheral blood, and PEC from rats were suspended in RPMI 1640 medium and cells were
infected at multiplicities of 0.1 f.f.u./cell for human mononuclear cells and 0.5 f.f.u./ceU for PEC of rats. After
incubation for 1 h at 37 °C for adsorption, ceils were washed twice with RPMI 1640 medium, resuspended in
RPMI 1640 medium supplemented with 10~ FCS and cultured in 24-well plastic plates in a CO2 incubator.
In]ectious centre assay. Inoculated cells were harvested at the day indicated until 2 weeks after virus inoculation,
and infectiouscentre assayswere performed. Seriallydiluted cell suspensionswere inoculated onto Vero E6 cells in
Lab-Tek 8 chambers. After adsorption for 1 h at 37 °C, maintenance medium containing 1.3~ methyl cellulose '
was overlaid. Direct IF tests were performed 7 days after infection as described above. At the same time, the
number of living cells were counted by the exclusion test, using 0.2~ (w/v) trypan blue dye.
Detection of viral antigen in PEC infected with B-1 strain virus. In order to determine the antigen-positive cells in
PEC at various days after infection, adherent cells were treated with PBS containing 0.2 ~ EDTA and 5 ~ FCS to
detach them from the plates, suspended in medium RPMI 1640 supplemented with 10~ FCS and seeded onto 12well spot-slides. PEC on slides were fixed and stained as described above, and the ratio of positive cells to total
cells was determined by fluorescence microscopy.
Antibody test by IF. For antibody detection, cells infected with the B-1 strain were fixed and stained by the
indirect IF test as described previously (Yamanishi et al., 1983).
RESULTS
Virus isolation f r o m newborn rats
Newborn rats were inoculated i.p. with the B-t strain. Virus isolation from brain, spleen,
lung, kidney and peripheral blood was attempted as described in Methods. Rats became ill, with
signs such as ruffled hair and hyper-excitability by 30 to 40 days after infection and some of them
died (data not shown). Virus could be isolated from all samples repeatedly from 1 week to at least
25 weeks post-infection, and antibody titres measured by IF started to increase at 3 weeks and
reached a m a x i m u m at 5 weeks post-infection (Table 1).
Next, rats infected with the B-1 strain were sacrificed at 5 weeks after infection and virus
isolation was attempted from various fractions of rat peripheral blood. As shown in Table 2,
virus could be isolated from the macrophage fraction (5.5 x 102 f.f.u./10 s cells) and the
granulocyte fraction (2.3 f.f.u./10 s cells). Approximately 0 . 5 5 ~ of the cells in the macrophage
population were virus-infected, a proportion about 240 times higher than that of infected cells
found in the granulocyte fraction. These data suggest that the virus would be highly associated
with the macrophage fraction in vivo,
Virus growth in P E C o f rats
Since the virus could be isolated mainly from macrophages of the peripheral blood of rats as
described above, virus growth in PEC was tested. Cells were infected with the B-1 strain at a
m.o.i, of 0,5 f.f.u./cell. Virus titres in supernatants of infected cells, the n u m b e r of infectious
centres and the n u m b e r of cells having viral antigen were calculated from 1 day to t4 days after
infection. As shown in Fig. 1, virus grew well in PEC. The m a x i m u m titres by infectious centre
assay and in the supernatant were 2.7 x 104 f.f.u./10 s cells and 4.9 x 104 f.f.u./ml respectively at
5 days after virus inoculation. The percentage of antigen-positive cells in PEC reached almost
100~ at 5 days after inoculation. The fluorescence pattern in PEC is shown in Fig. 2.
H F R S virus replication in human peripheral mononuclear cells
Mononuclear cells from h u m a n peripheral blood were fractionated into non-adherent
(consisting mainly of lymphocytes) and adherent cells (macrophages). A portion of the cell
suspension was treated with P H A - P and Lymphocult-T for lymphocytes, or with LPS for
macrophages, and infected with the B-1 strain at a m.o.i, of 0.1 f.f.u./cell. Virus titres in
supernatants and infectious centres were measured on Vero E6 cells. Virus did not multiply
appreciably in either non-activated or activated lymphocytes (Fig. 3a). In contrast, virus
replication was observed in macrophage-rich adherent cells which were either unstimulated or
LPS-stimulated (Fig. 3b). The m a x i m u m titre of virus was 7.1 f.f.u./105 cells by infectious centre
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 18:10:33
1274
T. N A G A I AND O T H E R S
Table 1. Virus isolation from various organs and peripheral blood of rats inoculated with B-1
strain i.p.*
Virus isolation from
Week after
virus
inoculation
1
3
5
8
11
15
20
25
c
~'
Brain
++
++
++
Lung
++
-~- -~++
Spleen
++
q-q++
Kidney
++
-Jr+
++
Peripheral
blood
++
NT~
++
Antibody
titre (4~)t
2
5
7
++
++
++
++
++
++
++
++
++
++
++
++
++
+
++
++
++
++
++
++
++
++
++
++
++
7
7
7
7
7
* Minced organs and peripheral blood from two rats were put onto Vero E6 cells. Inoculated cells were tested by
IF after 7 days or 14 days (passaged once) after inoculation. The positive rate was scored from - to + + ; positive
at 7 days after inoculation ( + +), 14 days after inoculation ( + ) .
t Antibody titres were measured by indirect IF as described in Methods.
NT, Not tested.
Table 2. Virus &olation from various fractions of peripheral blood of infected newborn rats at-5
weeks after inoculation
Fraction
Lymphocytes
Macrophages
Granulocytes
Erythrocytes
Virus titre
(f.f.u./105 cells)
<1
5.5 x 10z
2.3
<1
* Newborn rats were inoculated with HFRS virus B-1 strain within 24 h after birth. Virus isolation was
performed at 5 weeks after inoculation. Fractions were separated as described in Methods.
assay and 2-5 × 10 f.f.u./ml in supernatants at 10 days after inoculation. By activation with LPS,
virus growth seemed to be slightly enhanced, and the m a x i m u m titre rose to 5-9 × 10 per 105
cells by infectious centre assay and 3.0 × 102 f.f.u./ml in supernatants at 9 days after virus
inoculation (Fig. 3b). These results suggest that H F R S virus will grow also in human
macrophages in vivo.
DISCUSSION
H F R S virus usually infects man by the air-borne route or by rodent bite, and systemic
infection occurs. The major clinical manifestations are fever, prostration, proteinuria,
haemorrhagic phenomenon and renal failure (Lee, 1982). W h e n suckling mice were inoculated
i.p. or intracerebrally with H a n t a a n virus, virus antigen was present in almost all organs
including brain, spleen and lung (Kurata et al., 1983). In this paper, an H F R S - r e l a t e d virus (the
B-1 strain), which was isolated from a laboratory rat, was inoculated into newborn rats instead of
mice. Rats also manifested clinical symptoms such as ruffled hair and hyper-excitability as in
the case of mice, and virus could be isolated from brain, spleen, lung, kidney and peripheral
blood from 1 week to at least 25 weeks post-infection (Table 1). This evidence suggests that
the infection in rats persisted in some organs for at least 25 weeks and moreover viraemia
persisted for a long time in spite of the existence of high titred antibodies in the blood. It would
be interesting to examine in which fraction of blood cells the virus exists persistently. As shown
in Table 2, virus could be isolated from adherent cells (mainly macrophages) and virus growth in
vitro in PEC o f rats was considerable (Fig. 1). Since the purity o f the macrophages in PEC was
almost complete and the percentage of infected cells was close to I00%, it was confirmed that a
HFRS-related virus (the B-1 strain) had an affinity for the macrophages of rat.
When virus replication in human leukocytes was compared with that in rat macrophages from
peritoneal exudates, limited replication was observed only in the macrophage fraction from
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 18:10:33
H F R S virus in rats
I
~
I
I
I
1275
[
5
q00 o~"
31
Y,
50 :~
"*~ 2c
> 1
0
,
5
,
7
1
14
Time after infection (days)
Fig. 1. Virus replication in PEC from rats. Cells were infected with the B-1 strain at an m.o.i, of 0-5
f.f.u./cell. After adsorption for 1 h at 37 °C, the monolayers were washed twice with RPMI 1640
medium, overlaid with maintenance medium and incubated at 37 °C as described in Methods. Cultures
were harvested at the times indicated and virus titres were measured on Vero E6 cells. O, Virus fiti:e in
supernatant (f.f.u./ml); O, infectious centre assay (f.f.u./105 cells);/k, numbers of antigen-pos~i've cells
(%).
1
3
10
Fig. 2. Immunofluorescence staining of PEC infected with the B-1 strain. Cells were infected with the
B-1 strain, fixed 7 days after infection and stained by the direct IF method using monoclonal antibody
clone 80 conjugated with FITC. (a) Infected cells; (b) uninfected cells. Note the defined granular dots in
the cytoplasm.
human peripheral blood and virus growth was not significant in the lymphocyte fraction (Fig. 3).
It is known from several studies that mitogen-stimulated lymphocytes are permissive for the
replication of herpes simplex virus ( N a h m i a s et al., 1964; Westmoreland, 1978; Braun et al.,
1984), poliovirus (Willems et al., 1969), measles virus (Joseph et al., 1975; Sullivan et al., 1975)
and dengue virus (Theofilopoulos et al., 1976), and also that dengue virus could multiply in
cultures of mouse peritoneal macrophages activated with bacterial LPS (Hotta & Hotta, 1982).
In this paper, stimulated lymphocytes or macrophages were infected with the B-1 strain of
H F R S virus. While virus growth in LPS-stimulated macrophages was enhanced, no viral
replication was observed in P H A - s t i m u l a t e d lymphocytes (Fig. 3). These d a t a suggest that virus
replication is dependent on the state of cell differentiation.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 18:10:33
1276
4
T. N A G A I AND OTHERS
I
I
I
I
I
I
I
I
I
I I . . . . . . .
1
I
I
I
I
•
, ,
,
,
l
i
I
I
|
I
I
I
I
I
I
I
I
I
I
~
-E 3'
~2
.~.
1
1
3
5
7
9
11
13
0 1
3
Time after infection (days)
5
7
9
11
13
Fig. 3. Growth curves of the B-1 strain in human mononuclear cells. (a) Lymphocytes were either
stimulated with PHA-P (lower panel) or not (upper panel); (b) macrophages were either stimulated with
LPS (lower panel) or not (upper panel) and infected with the B-1 strain and at each time point, samples
of culture fluid or infected cells were harvested. The infectivity yield in fluid (O) (f.f.u./ml) and the
numbers of infected cells ((3) (f.f.u./10 5 cells) were determined as described in Methods.
It is evident that some viruses multiply in certain leukocyte fractions (Bang & Warwick, 1960;
Goodman & Koprowski, 1962; Edelman & Wheelock, 1967; Wheelock & Edelman, 1969;
Willems et al., 1969; Joseph et al., 1975; Sullivan et aL, 1975; Rinaldo et al., 1978;
Theofilopoulos et al., 1976; Turner & Ballard, 1976; Halstead et al., 1977; Levitt et al., 1979).
These observations are interesting when we consider the mechanism responsible for systemic
infection. Even though the macrophage might not be a primary target of HFRS virus, it is
conceivable that systemic infection with HFRS virus could be caused by macrophages which
might act as a carrier or propagator of the virus. It has been reported that viral antigen is present
in endothelial cells lining blood vessels (Kurata et al., 1983). If the virus propagates in
macrophages, infected macrophages could attach to the endothelial cells and consequently virus
could be systemically spread to various organs. Moreover, it would also be possible for virus to
pass the blood-brain barrier and to infect the brain. In fact, virus was isolated from the brain as
early as the seventh day post-infection as shown in Table 1.
HFRS is a systemic disease and the antibody titres in sera of patients or infected animals were
extremely high. Recently, the mechanism of prevention and recovery from viral diseases have
been extensively studied with respect to both humoral immunity and cell-mediated immunity,
including cytotoxic T cells, natural killer activity and delayed-type hypersensitivity (DTH).
Macrophages have an important role in host defence mechanisms as phagocytic cells, as the
effector cells in antibody-dependent cytotoxicity and in DTH, and also as the cells that produce
interferon (Hirsch, 1984). If macrophages are incapacitated by viral infection, it would be
expected that other defence mechanisms such as high titred antibodies and cytotoxic T cells will
work cooperatively towards recovery from disease. The mechanisms for recovery from HFRS
virus infection in animals are under investigation.
REFERENCES
ALLlSON, A. C., DAVrES,P. & PACE, R. C. (1973). Effects of endotoxin on macrophages and other lymphoreticular
cells. Journal of Infectious Diseases (supplement 128), 212-219.
BANO, F. 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, U.S.A. 46, 1065-1075.
BRAUN, R . W . , TEUTE, H. K., KIRCHNER, H. & MUNK, K. (1984). Replication of herpes simplex v i r u s i n h u m a n T
lymphocytes: characterization of the viral target cell. Journal oflmmunology 132, 914 919.
CASALS, J., HOOGSTRAAL, H., JOHNSON, K. M., SHELOKOV, A., WIEBENGA, N. H. & W O R K , T. (1966). A c u r r e n t appraisal
of hemorrhagic fever in the U.S.S.R. American Journal of Tropical Medicine and Hygiene 15, 751-764.
COHEN, M. S., CASALS, J., HSIUNG, D-G., KWE1, H., CHIN, C., GE, H., HSIANG, C., LEE, P. W., GIBBS, C. J., JR & GAJDUSEK,
D. C. (1981). Epidemic haemorrhagic fever in Hubei province, the People's Republic of China: a clinical and
serological study. Yale Journal of Biology and Medicine 54, 41-55.
DESMYTER, J., LEDUC, J. W . , JOHNSON, K. M., BRASSEUR, F., DECKERS, C. & VAN YPERSELE STRIHOU, C. (1983).
Laboratory rat associated outbreak of haemorrhagic fever with renal syndrome due to Hantaanqike virus in
Belgium. Lancet ii, 1445-1448.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 18:10:33
H F R S virus in rats
1277
EDELMAN, R. & WHEELOCK,E. F. (1967). Specific role of each h u m a n leukocyte type in viral infections. I. Monocyte
as host cell for vesicular stomatitis virus replication in vitro. Journal of Virology 1, 1139 1 !,49.
GOODMAN,G. T. & KOPROWSKI,a. (1,962). Study of the m e c h a n i s m of innate resistance to virus infection. Journal of
Cellular Physiology 59, 333-373.
HALSTEAD,S. B., O'ROURKE, E. J. & ALLISON,A. C. (1977). Dengue viruses and mononuclear phagocytes, lI. Identity
of blood and tissue leukocytes supporting in vitro infection. Journal of Experimental Medicine 146, 218229.
HIRSCH, M. S. (t984). Pathogenesis of viral infections. In Antiviral Agents and Viral Diseases of Man, 2nd edn., pp.
35--54. Edited by G. J. Galasso, T. C. Merigan & R. A. Buchanan. New York: R a v e n Press.
HOTTA, H. & HOTTA,S, (1982). Dengue virus multiplication in cultures of mouse peritoneal macrophages: effects of
macrophage activators. Microbiology and Immunology" 26, 665-676.
ISHII, S., ANDO,K., WATANABE,N., MURAKAMI,R., NAKAYAMA,T. & ISHIKAWA,I. (1942). Studies on Songo fever. Japan
Army Medical Journal 355, 1755-1758 (in Japanese).
JOSEPH, B. S., LAMPERT,P. W. & OLDSTONE,M. B. A. (1975). Replication and persistence of measles virus in defined
subpopulations of h u m a n leukocytes. Journal of Virology 16, 1,638-1649.
KAWAMATA, J. (1983). Studies on the Surveillance and Control Measures of Zoonoses Associated with Animal
Experimentation with Special Reference to Hemorrhagic Fever. A report from the Ministry of Education,
Cultural Affairs and Science (in Japanese).
KITAMURA,T., MORITA,C., KOMATSU,T., SUGIYAMA,K., ARIKAWA,J., SHIGA, S., TAKEDA,H., AKAO,Y., IMAIZIMI, K.,
OYA, A., HASHIMOTO, N. & URASAWA, S. (1983). Isolation of virus causing hemorrhagic fever with renal
syndrome (HFRS) through a cell culture system. Japanese Journal of Medical Science and Biology 36, 17-25.
KUMAGAI,K., ITOH, K., HINUMA, S. & TADA, M. (1979). Pretreatment of plastic Petri dishes with fetal calf serum. A
simple method for macrophage isolation. Journal oflmmunological Methods 29, 17 25.
KURATA, T., TSAI, T. F., BAUER, S. P. & McCORMICK, J. B. (1983). Immunofluorescence studies of disseminated
H a n t a a n virus infection of suckling mice. Infection and Immunity 41, 391-398.
LAEDVlRTA, J. (1971). Nephropathia epidemica in Finland. Annals of Clinical Research 3, 12-17.
LEE, H. W. (1982). Korean hemorrhagic fever. Progress in Medical Virology 28, 96-113.
LEE, H. W., LEE, P. W. & JOHNSON,K. M. (t978). Isolation of etiologic agent of Korean hemorrhagic fever. Journal of
Infectious Diseases 137, 298--308.
LEE, H. w., BALK, L. J. & JOHNSON, K. M. (1982a). Isolation of H a n t a a n virus, the etiologic agent of Korean
hemorrhagic fever, from wild urban rats. Journal of Infectious Diseases 146, 638-644.
LEE, P. w., AMYX, H. L., GAJDUSEK, D. C., YANAGIHARA,R. T., GOLDGABER, D. & GIBBS, C. J., JR (1982b). New
hemorrhagic fever with renal syndrome-related virus in indigenous wild rodents in the United States. Lancet
ii, 1405.
LEVITT, N. H., MILLER,H. V. & EDELMAN,R. (1979). Interaction of alphaviruses with h u m a n peripheral leukocytes: in
vitro replication of Venezuelan equine encephalitis virus in monocyte cultures. Infection and Immunity 24,
642-646.
MERY, J. P., DARD, S., CHAMOUARD,J. M., DOURNON, E., BRICAIRE, F., VAHERI, A., BRUMMER-KORVENKONTIO,M.,
GONZALEZ, J. P. & McCORMICK, J. B. (1983). Muroid virus nephropathies. Lancet ii, 845-846.
MYHRMAN, G. (1951). Nephropathia epidemica, a new infectious disease in Northern Scandinavia. Acta medica
scandinavica 140, 52-56~
NAHMIAS, A. J., KmRICK, S. & ROSAN, R. C. (1964). Viral leukocyte interrelationships. I. Multiplication of a D N A
virus - herpes simplex - in h u m a n leukocyte cultures. Journal of Immunology 93, 69-74.
NIKLASSON, B. & LEDUC, J. (1984). Isolation of the nephropathia epidemica agent in Sweden. Lancet i, 1012-1013.
RINALDO, C. R., JR, RICHTER, B. S., BLACK,P. H., CALLERY,R., CHESS, L. & HIRSCH, M. S. (1978). Replication of herpes
simplex virus and cytomegalovirus in h u m a n leukocytes. Journal of lmmunology 120, 130-136.
SMADEL, J. E. (1953). Epidemic hemorrhagic fever. American Journal of Public Health 43, 1327-1330.
SONG, G., HANG, C., QUI, X., NI, D., LIAO, H., GAO, G., DU, Y., XU, J., WU, Y., ZHAO, J., KONG, B., WANG, Z., ZHANG, Z.,
SHEN, H. & ZHOU, N. (1983). Etiologic studies of epidemic hemorrhagic fever (hemorrhagic fever with renal
syndrome). Journal of lnfectious Diseases 147, 654-659.
SULLIVAN,J. L., BARRY,D. w., LUCAS,S. J. & ALBRECHT,P. (1975). Measles infection of h u m a n mononuclear cells. I.
Acute infection of peripheral blood lymphocytes and monocytes. Journal of Experimental Medicine 142, 773784.
THEOFILOPOULOS, A. N., BRANDT, W. E., RUSSELL, P. K. & DIXON, F. T. (1976). Replication of dengue-2 virus in
cultured h u m a n lymphoblastoid cells and subpopulations of h u m a n peripheral leukocytes. Journal of
Immunology 117, 953-961.
TURNER, G. S. & BALLARD,R. (1976). Interaction of mouse peritoneal macrophages with fixed rabies virus in vivo
and in vitro. Journal of General Virology 30, 223-231.
WESTMORELAND, D. (1978). Herpes simplex virus type-1 and h u m a n iymphocytes: virus expression and the
response to infection of adult and foetal cells. Journal of General Virology 40, 559-575.
WHEELOCK, E. F. & EDELMAN,R. (1,969). Specific role of each h u m a n leukocyte type in viral infections. III. 17D
yellow fever virus replication and interferon production in homogeneous leukocyte cultures treated with
phytohemagglutinin. Journal of Immunology 103, 429-436.
WILLEMS,F. T. C., MELNICK,J. L. & RAWLS,W. E. (1969). Replication of poliovirus in phytohemagglutinin-stimulated
h u m a n lymphocytes. Journal of Virology 3, 451-457.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 18:10:33
1278
T. N A G A I
AND
OTHERS
YAMANISHI, K., DANTAS, F. J. R., TAKAHASHI, M., YAMANOUCHI, T., DOMAE, K., KAWAMATA, J. & KURATA, T. (1983).
Isolation of hemorrhagic fever with renal syndrome (HFRS) virus from a tumor specimen in a rat. Biken
Journal 26, 155-160.
YAMANISHI,K., DANTAS,J. R., JR, TAKM-I~HI, M., YAMANOUCHI,T., DOMAE,K., TAKM-IASHI,Y. & TANISHITA,O. (1984).
Antigenic differences between two viruses, isolated in Japan and Korea, that cause hemorrhagic fever with
renal syndrome. Journal of Virology 52, 231-237.
YANAGIHARA, R., GOLDBABER, D., LEE, P. W., AMYX, H. L., GAJDUSEK, D. C. & GIBBS, J. C., JR (1984). P r o p a g a t i o n o f
nephropathia epidemica virus in cell culture. Lancet i, 1013.
(Received 20 November 1984)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 18:10:33