Pathology of Puumala Hantavirus Infection in Macaques
Tarja Sironen1*, Jonas Klingstro ¨m2, Antti Vaheri1, Leif C. Andersson3, A˚ke Lundkvist2, Alexander
1Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland, 2Swedish Institute for Infectious Disease Control, and MTC, Karolinska Institutet,
Stockholm, Sweden, 3HUSLAB and Department of Pathology, Haartman Institute, University of Helsinki, Helsinki, Finland
Hantaviruses are globally important human pathogens that cause hemorrhagic fever with renal syndrome and hantavirus
pulmonary syndrome. Capillary leakage is central to hantaviral diseases, but how it develops, has remained unknown. It has
been hypothesized that the pathogenesis of hantavirus infection would be a complex interplay between direct viral effects
and immunopathological mechanisms. Both of these were studied in the so far best model of mild hemorrhagic fever with
renal syndrome, i.e. cynomolgus macaques infected with wild-type Puumala hantavirus. Viral RNA detected by in situ
hybridization and nucleocapsid protein detected by immunohistochemical staining were observed in kidney, spleen and
liver tissues. Inflammatory cell infiltrations and tubular damage were found in the kidneys, and these infiltrations contained
mainly CD8-type T-cells. Importantly, these results are consistent with those obtained from patients with hantaviral disease,
thus showing that the macaque model of hantavirus infection mimics human infection also on the tissue level. Furthermore,
both the markers of viral replication and the T-cells appeared to co-localize in the kidneys to the sites of tissue damage,
suggesting that these two together might be responsible for the pathogenesis of hantavirus infection.
Citation: Sironen T, Klingstro ¨m J, Vaheri A, Andersson LC, Lundkvist A˚, et al. (2008) Pathology of Puumala Hantavirus Infection in Macaques. PLoS ONE 3(8):
Editor: Douglas F. Nixon, University of California San Francisco, United States of America
Received July 8, 2008; Accepted July 30, 2008; Published August 21, 2008
Copyright: ? 2008 Sironen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from The Academy of Finland, Helsinki Biomedical Graduate School, Sigrid Juse ´lius Foundation, Finnish Cultural
Foundation, and from the Swedish Medical Research Council. None of these had any role in the study.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Tarja.Sironen@helsinki.fi
Hantaviruses (genus Hantavirus, family Bunyaviridae) are rodent-
borne viruses that have a tripartite genome of negative-strand
RNA . They are the causative agents of hemorrhagic fever with
renal syndrome (HFRS) in Eurasia, and hantavirus pulmonary
syndrome (HPS) in the Americas. A mild form of HFRS called
nephropathia epidemica (NE) is caused by Puumala virus (PUUV)
. Although capillary leakage is the hallmark of hantaviral
diseases, its detailed mechanisms remain largely unknown.
Hantaviruses commonly infect endothelial cells , which is
thought to play a key role in the development of HFRS and HPS.
Increased levels of serum perforin, granzyme B, and caspase-
cleaved cytokeratin-18 have been reported in HFRS patients,
suggesting some tissue damage . Yet, direct viral cytotoxicity is
unlikely to be the primary cause of pathology: hantaviruses are not
cytopathic in vivo , and hantavirus infection alone does not affect
the permeability of endothelial cells in vitro . A second factor
might be the host immune response. CD8+ T cells can in vitro
increase the permeability of human endothelial cell monolayers 
and high levels of these cells are observed during acute HPS .
Furthermore, immunoblasts consisting largely of CD8+ T cells are
detected both in the lungs of HPS patients [3,8] and in the kidneys
of NE patients . Additionally, tissue damage could be caused by
the overproduction of cytokines by infected monocyte/macro-
phages, especially TNF-a that is known to increase vascular
permeability. Increased levels of the cytokines TNF-a, IL-6, and
IL-10 have been reported in NE patients [10,11] Furthermore,
high levels of cytokine-producing cells are seen in the lungs of HPS
patients , and the pulmonary fluid of HPS patients appears to
be exudative in nature .
The role of different factors in hantavirus pathogenicity is best
evaluated in a model system. In their natural hosts, hantaviruses
cause an asymptomatic and persistent infection with no apparent
pathology, and therefore, the use of rodent-based models is
limited. Syrian hamsters infected with either Andes (ANDV) or
Maporal virus do develop a disease similar to HPS, while another
HPS-causing hantavirus, Sin Nombre, infects the hamsters
asymptomatically [14–17]. HFRS hantaviruses are non-pathogen-
ic to hamsters even at very high doses, and although suckling mice
can be infected by HFRS hantaviruses, the disease does not
resemble the disease in humans . The first attempts to establish
a monkey-based model were not successful either, probably due to
the use of cell culture-adapted virus. The adaptation of wild-type
PUUV (passaged in colonized bank voles) to cultured primate
Vero E6 cells leads to genetic and phenotypic changes [19,20]: the
cell culture–adapted variant is noninfectious to bank voles. Many
species of nonhuman primates develop an antibody response to
PUUV or Prospect Hill virus (PHV) infection, but lack disease
. ANDV infection of macaques fails to cause in any disease,
although there is an antibody response . PUUV (cell culture –
adapted) infection results in some mild symptoms, but without
inflammatory reaction in the kidneys, which is in contrast to
human NE-cases . Cynomolgus macaques (Macaca fascicularis)
infected with wild-type PUUV (strain Kazan-wt, passaged in
colonized bank voles), however, develop a disease that mirrors
closely NE in humans , including renal involvement and
elevated cytokines. This model has been used successfully to study
PLoS ONE | www.plosone.org1August 2008 | Volume 3 | Issue 8 | e3035
the protective potential of passive immunization against hantavirus
infection . Here, we present the histopathological examination
of three monkeys described previously , focusing on
characterization of the distribution of PUUV in tissues, and
involvement of cytotoxic T-cells (CTLs).
All the three PUUV-infected monkeys lost their appetite during
days 7 to 14 after the infection. During this period they were
apathetic, and increased temperature was clearly demonstrated for
one of the monkeys (#59) . Similarly to human patients, the
severity of the symptoms ranged from mild to severe. Kidney
involvement was apparent since two of the monkeys developed
proteinuria and hematuria. Nitric oxide responses were observed
in all three monkeys, and elevated values of CRP and creatinine in
two of the monkeys. Increased level of the cytokine IL-6 was seen
in all three monkeys, and increased TNF-a in two monkeys .
In this study, we first analyzed the presence and location of
PUUV genomes in tissue samples using in situ hybridization. The
specificity of the probe was initially confirmed using Vero E6 cells:
PUUV-infected cells as a positive control (Fig. 1A), and mock-
infected cells as a negative control (Fig. 1B). No signal was
observed in mock-infected cells. These cells were also stained with
the polyclonal anti-PUUV-N antibody  to confirm the
infection (Fig. 1C and 1D). PUUV RNA (negative strand) was
detected in kidney (Fig. 2A), spleen (Fig. 2B) and liver (Fig. 2C)
tissues of the infected macaques. These tissues were earlier found
RT-PCR positive for PUUV RNA . Tissue samples from a
non-infected control monkey had no detectable signal (Fig. 2D–F).
All monkeys had PUUV RNA in the kidneys, and the most
severely affected monkey #59 gave the strongest signal (Fig. 2A,
Table 1). Importantly, PUUV RNA was mostly seen in distal
tubuli, and also in the lumen of the tubuli suggesting secretion of
the virus into urine. In addition to kidney tubular epithelial cells,
viral markers were occasionally found within capillary endothe-
lium in kidney, liver and spleen. In the liver samples of two
monkeys (#59 and #53) the virus was mostly found in Kupfer
cells (Fig. 2C). In the spleen samples of these two monkeys, PUUV
RNA was also detected, and the signal was mostly endothelial
(capillaries) with some positive dendritic cells (Fig. 2B). The
infected cells were identified based on the morphological
characteristics of the cells. The heart and lung tissues remained
negative in the in situ hybridization, although all the heart samples
and one lung sample (from monkey #59) were RT-PCR positive
. In general, PUUV RNA was detected focally and at a low
level in the tissues (except for the kidneys), and thus, the tissue
stainings on a few sections might miss some of the positive foci.
infiltrates mainly in the kidneys (Fig. 3), but some also in the
lungs, and the heart. The tissue samples were collected 28 days
after the infection, and it seemed that, at this time point, the viral
replications had already ceased in the lungs and the heart, but the
inflammation had not resolved fully yet. In the kidneys the
inflammatory cell infiltrates were mostly tubulointerstitial, and
while the infiltrates were absent in the least affected monkey #25,
they were clearly detectable in monkeys #53 and #59. In the
lungs interstitial pneumonia, thickening of alveolar septa and
scattered lymphocytes were seen. Mild myocarditis was detected,
while the liver and spleen appeared normal.
The number of the inflammatory cells was evaluated on an
arbitrary scale. The inflammatory cells were predominantly CD3-
positive T-cells, (Fig. 4A, Table 1), and a large proportion of these
were of CD8-type (Fig. 4B). Staining was particularly pronounced
at the sites of damaged tubuli (Figure 3, magnification). The
highest amount of inflammatory cell infiltrates was detected in
monkey #53. Notably, serially cut sections showed that PUUV
RNA and N protein were detected at similar sites of tubular
damage (Fig. 4C and D). Taken together, these results implied that
the immunopathology caused by T-cells could be provoked
directly by PUUV replication. Furthermore, the level of disease
severity and the amount of virus detected in the infected macaques
(Table 1) seemed to correlate. The least affected monkey (#25)
was weakly positive for PUUV and only in the kidneys, where no
inflammation above background was observed. Monkeys #53 and
#59, which had clear clinical symptoms, also showed high level of
inflammatory cell infiltrates and PUUV, especially in the kidneys.
The difference in the distribution of viral markers and inflamma-
tory cells in these two monkeys could be due to individual response
and different kinetics of the infection.
In agreement with the current hypothesis on pathogenesis of
hantavirus infection, both the virus and T-cells were detected in
the two apparently ill macaques at the site of tissue damage.
Autopsy data on human HPS cases caused by Sin Nombre virus
infection have revealed that hantaviral antigens are mostly found
in endothelial cells, but also other types of cells are positive, such as
monocyte/macrophages . PUUV antigen has been detected in
the endothelium of the pituitary gland and in kidney tubuli of NE
patients . In one study on human kidney biopsies , PUUV
antigen was detected in the cytoplasm of renal tubular epithelial
cells with focal distribution in the cortical and medullary areas of
the kidney. It therefore seems that in our study, PUUV RNA and
antigen were detected in the monkey tissues in the same cell types
and locations with focal distribution as in human autopsy tissue
samples, confirming that Cynomolgus macaques infected with wt-
PUUV mimic hantavirus disease in humans. Interestingly, the
severity of human HFRS-cases ranges from mild (sometimes
almost symptomless) to severe and to lethal, and similarly did the
severity of the symptoms and pathological findings range also in
the macaques. The important feature of this macaque model in
Figure 1. Specificity of the in situ hybridization probe. PUUV RNA
was detected by in situ hybridization, and visualized with diamino-
benzidine in PUUV-infected Vero E6 cells (A), while mock-infected cells
(B) showed no signal. PUUV N antigen was detected by immunohis-
tochemistry, visualized by diaminobenzidine, in PUUV-infected cells (C),
while no staining was seen in mock-infected cells (D).
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comparison to other animal models for hantaviral diseases, is the
finding of both the virus and the inflammatory cell infiltrates: the
latter are absent in the naturally infected rodents hosts as well as in
the earlier attempted macaque models [21–23]. Furthermore, the
Syrian hamster model that works well with the HPS-causing
ANDV  is apparently not applicable to HFRS-causing
hantaviruses, which makes this macaque-model, at the moment,
even more valuable.
Immunopathology is critical in hantaviral diseases. The HLA-
B*3501 haplotype is associated with severe HPS caused by SNV
infection implying involvement of CD8+ CTLs, and the number
of SNV-specific CD8+ T cells correlates with disease severity .
Similarly, HLA-B8-DR3 is associated with severe outcome of
PUUV infection , and the PUUV RNA level in such patients
was particularly high, suggesting impaired handling of infection
. In general, this haplotype affects the early stages of immune
activation by altering the balance of cytokines produced .
Furthermore, in kidney biopsies of NE patients the typical
histopathological finding is acute tubulointerstitial nephritis, and
inflammatory cell infiltrations . These focal immunoblasts are
similarly seen in the macaque model and they consist mainly of
CTLs (Fig. 1D). In addition to T-cells, these infiltrations included
some CD45- and CD20-positive B-cells (Table 1) in the monkey
In NE patients, the CD8+ T cell response peaks at the onset of
clinical disease, and decreases gradually within the next three
weeks . When the peak effector response subsides, the memory
T cells start to emerge during the first weeks of convalescence.
Thus, in our monkey tissue samples taken approximately 2–3
weeks after the onset of symptoms, the T cell response is already
decreasing, and yet we find a high level of T-cell infiltrates, at least
in the two more severely affected monkeys.
In general, the tissue samples from the PUUV-infected monkeys
and from NE patients, have surprisingly low level of tissue damage
despite clearly increased capillary leakage. It is known that
Figure 2. Detection of PUUV genome. PUUV S segment RNA was detected by in situ hybridization, and visualized with Ventana Blue in the
kidney (A), spleen (B), and liver (C) of the monkey #59. In the spleen, PUUV RNA signal was mostly endothelial (capillaries) with some positive
dendritic cells (B), and in the liver samples PUUV RNA was found in Kupfer cells (C). Scale bar corresponds to 50 mm. Negative control tissues were
stained in parallel to the infected monkeys. No signal was detected in the kidney (D), spleen (E), or liver (F) of the negative control monkey.
Table 1. Detection of Puumala virus RNA and inflammatory
cells in tissue samples
A: Puumala virus RNA (in situ hybridization*)
B: Inflammatory cells** (in kidneys)
the severity of infection increases from left to right
*RT-PCR amplification of PUUV RNA was positive for all blood and tissue
samples, except for the lung tissue samples of the monkeys #25 and #53
**Antigen scoring: 2=no positive cells, +=rare positive cells, ++=moderate
number of positive cells, +++=many positive cells
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Figure 3. HE staining shows lymphoid cells (arrows) in the kidney tissue of the monkey #53. Scale bar corresponds to 500 mm. The
magnification shows a damaged tubulus surrounded by inflammatory cells.
Figure 4. Characterization of the focal inflammatory cell infiltrates. These consist mainly of T-cells that are stained immunohistochemically
with a polyclonal anti-CD3 antibody (A), and are visualized with Vector Red. A proportion of these T-cells is also positive for T-cell intracellular
antigen-1 (TIA-1), a marker for active cytotoxic T cells (B). PUUV RNA, visualized with Ventana Blue (C), and PUUV N antigen, detected with a
polyclonal anti-PUUV-N antibody, and visualized with diaminobenzidine (D), are found at the site of infiltrates in the kidneys of the monkey #53.
Scale bars correspond to 100 mm (A and C) and 25 mm (B and D).
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hantavirus infection can increase the permeability of human
endothelial cells in vitro by subtle mechanisms, and before apparent
cytopathic effects . Furthermore, hantavirus-specific CTLs can
increase the endothelial cell permeability after antigen-recognition.
It has been speculated that the mechanism of immunopathogen-
esis could be similar to the interactions between endothelial cells
and CD8+ T-cells . Normally, endothelial cells have higher-
than-average tolerance for the cytolytic attack of CD8+ T-cells,
and this down-regulation is mediated via binding of programmed
death 1 –receptor (PD-1) on CTLs to the ligand PD-L1/L2
expressed on endothelial cells. An intriguing possibility is that in
hantavirus infection this interaction could be disturbed either via
excess amount of activated CTLs or alternatively, hantavirus
infections could modulate the endothelial cell functions for
example via changes in the expression of PD-L1/L2 .
Interestingly, renal tubular epithelial cells, another target of
PUUV, can also down-regulate specific T-cell responses by
increasing the expression of PD-L1 . In the monkey tissues,
hantavirus-infected endothelial and kidney epithelial cells were
found at sites of T-cell infiltration supporting the hypothesis of
virus-induced immunomodulation. This phenomenon, as well as
other key issues of the pathology of hantavirus infection, may now
be studied with the Cynomolgus macaque model.
Materials and Methods
The details of the experimental infection were described
earlier . The housing, maintenance and care of the animals
was performed according to the relevant guidelines and
requirements of the Swedish Institute for Infectious Disease
Control. Briefly, the monkeys were inoculated intravenously with
approximately 105bank vole 50% infective doses of PUUV strain
Kazan-wt  in 1 ml of phosphate-buffered saline. Lung tissue
material from infected bank voles was used as the inoculum.
After 28 days of infection, the animals were sacrificed, and tissue
Samples of lung, heart, spleen, liver and kidney were dissected
and stored frozen at 270uC. Tissues were fixed in 3%
paraformaldehyde for 48 hours, dehydrated and paraffin-embed-
ded. 4 mm sections were cut on slides. The sections were stained
with hematoxylin-eosin for standard histopathological analysis.
Immunohistochemical detection of CD20, CD45, CD3, and TIA-
1 was done using VectaStain ABC kit with Vector Red as
substrate. Detection of PUUV N protein was performed on the
fully automated Ventana Discovery Slide stainer. Sections were
first deparaffinized and pretreated in microwave oven in citrate
buffer pH 6.0. Polyclonal antibody against Puumala virus N
protein was used in 1:100 dilution. Ventana iViewDAB kit was
used for detection, and sections were counterstained with
hematoxylin and postcounterstained with Bluing Reagent. Finally,
the slides were rinsed and dehydrated before mounting with
EuKitt mounting medium.
In situ hybridization
A pcDNA3-plasmid containing PUUV S segment coding
sequence (nt 61–1261) was linearised with BglII restriction
enzyme. A probe hybridizing to nt 843–1261 of viral S segment
RNA was in vitro transcribed and labeled with digoxigenin (DIG
RNA labeling kit, Roche Applied Science). The specificity of the
probe was first confirmed on VeroE6-cells that were infected with
PUUV, strain Sotkamo, or mock-infected. In situ hybridization
was carried out on the automated stainer. Sections were
deparaffinized, fixed with RiboPrep and digested with Protease3.
250 ng of riboprobe was added to the slide and hybridized for
10 hours. Sections were washed, postfixed, endogenous biotin was
blocked and hybridized probes were detected with BlueMap kit.
Finally, slides were counterstained with nuclear fast red, rinsed,
dehydrated and mounted permanently with EuKitt mounting
medium. Tissue sections from a non-infected monkey were used as
negative controls for both immunohistochemistry and in situ
Irina Suomalainen is thanked for her expert technical assistance with
Conceived and designed the experiments: JK AV LCA L AP. Performed
the experiments: TS JK. Analyzed the data: TS LCA AP. Contributed
reagents/materials/analysis tools: AV LCA L AP. Wrote the paper: TS JK
AV LCA L AP.
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