Tacaribe virus but not junin virus infection induces cytokine release from primary human monocytes and macrophages.
ABSTRACT The mechanisms underlying the development of disease during arenavirus infection are poorly understood. However, common to all hemorrhagic fever diseases is the involvement of macrophages as primary target cells, suggesting that the immune response in these cells may be of paramount importance during infection. Thus, in order to identify features of the immune response that contribute to arenavirus pathogenesis, we have examined the growth kinetics and cytokine profiles of two closely related New World arenaviruses, the apathogenic Tacaribe virus (TCRV) and the hemorrhagic fever-causing Junin virus (JUNV), in primary human monocytes and macrophages. Both viruses grew robustly in VeroE6 cells; however, TCRV titres were decreased by approximately 10 fold compared to JUNV in both monocytes and macrophages. Infection of both monocytes and macrophages with TCRV also resulted in the release of high levels of IL-6, IL-10 and TNF-α, while levels of IFN-α, IFN-β and IL-12 were not affected. However, we could show that the presence of these cytokines had no direct effect on growth of either TCRV of JUNV in macrophages. Further analysis also showed that while the production of IL-6 and IL-10 are dependent on viral replication, production of TNF-α also occurs after exposure to UV-inactivated TCRV particles and is thus independent of productive virus infection. Surprisingly, JUNV infection did not have an effect on any of the cytokines examined indicating that, in contrast to other viral hemorrhagic fever viruses, macrophage-derived cytokine production is unlikely to play an active role in contributing to the cytokine dysregulation observed in JUNV infected patients. Rather, these results suggest that an early, controlled immune response by infected macrophages may be critical for the successful control of infection of apathogenic viruses and prevention of subsequent disease, including systemic cytokine dysregulation.
- SourceAvailable from: Olga A Kolokoltsova[Show abstract] [Hide abstract]
ABSTRACT: Junin virus (JUNV) is the etiological agent of Argentine hemorrhagic fever (AHF), a human disease with a high case-fatality rate. It is widely accepted that arenaviral infections, including JUNV infections, are generally non-cytopathic. In contrast, here we demonstrated apoptosis induction in human lung epithelial carcinoma (A549), human hepatocarcinoma and Vero cells upon infection with the attenuated Candid#1 strain of, JUNV as determined by phosphatidylserine (PS) translocation, Caspase 3 (CASP3) activation, Poly (ADP-ribose) polymerase (PARP) cleavage and/or chromosomal DNA fragmentation. Moreover, as determined by DNA fragmentation, we found that the pathogenic Romero strain of JUNV was less cytopathic than Candid#1 in human hepatocarcinoma and Vero, but more apoptotic in A549 and Vero E6 cells. Additionally, we found that JUNV-induced apoptosis was enhanced by RIG-I signaling. Consistent with the previously reported role of RIG-I like helicase (RLH) signaling in initiating programmed cell death, we showed that cell death or DNA fragmentation of Candid#1-infected A549 cells was decreased upon siRNA or shRNA silencing of components of RIG-I pathway in spite of increased virus production. Similarly, we observed decreased DNA fragmentation in JUNV-infected human hepatocarcinoma cells deficient for RIG-I when compared with that of RIG-I-competent cells. In addition, DNA fragmentation detected upon Candid#1 infection of type I interferon (IFN)-deficient Vero cells suggested a type I IFN-independent mechanism of apoptosis induction in response to JUNV. Our work demonstrated for the first time apoptosis induction in various cells of mammalian origin in response to JUNV infection and partial mechanism of this cell death.PLoS ONE 01/2014; 9(6):e99610. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Double-stranded RNA (dsRNA) is synthesized during the course of infection by RNA viruses as a byproduct of replication and transcription and acts as a potent trigger of the host innate antiviral response. In the cytoplasm of the infected cell, recognition of the presence of viral dsRNA as a signature of "non-self" nucleic acid is carried out by RIG-I-like receptors (RLRs), a set of dedicated helicases whose activation leads to the production of type I interferon α/β (IFN-α/β). To overcome the innate antiviral response, RNA viruses encode suppressors of IFN-α/β induction, which block RLR recognition of dsRNA by means of different mechanisms that can be categorized into: i) dsRNA binding and/or shielding ("hide"), ii) dsRNA termini processing ("mask") and iii) direct interaction with components of the RLRs pathway ("hit"). In light of recent functional, biochemical and structural findings, we review the inhibition mechanisms of RLR recognition of dsRNA displayed by a number of highly pathogenic RNA viruses with different disease phenotypes such as haemorrhagic fever (Ebola, Marburg, Lassa, Lujo, Machupo, Junin, Guanarito, Crimean-Congo, Rift Valley fever, dengue), severe respiratory disease (influenza, SARS, Hendra, Hantaan, Sin Nombre, Andes) and encephalitis (Nipah, West Nile).Antiviral research 10/2013; · 3.61 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Arenavirus Junin is the causative agent of Argentine hemorrhagic fever. Limited information is available concerning the pathogenesis of this human disease, especially the pathogenesis of acute and late neurological symptoms.Virology journal. 07/2014; 11(1):126.
Tacaribe Virus but Not Junin Virus Infection Induces
Cytokine Release from Primary Human Monocytes and
Allison Groseth1, Thomas Hoenen1, Michaela Weber1, Svenja Wolff1, Astrid Herwig1, Andreas
Kaufmann2, Stephan Becker1*
1Institut fu ¨r Virologie, Philipps Universita ¨t Marburg, Marburg, Germany, 2Institut fu ¨r Immunologie, Philipps Universita ¨t Marburg, Marburg, Germany
The mechanisms underlying the development of disease during arenavirus infection are poorly understood. However,
common to all hemorrhagic fever diseases is the involvement of macrophages as primary target cells, suggesting that the
immune response in these cells may be of paramount importance during infection. Thus, in order to identify features of the
immune response that contribute to arenavirus pathogenesis, we have examined the growth kinetics and cytokine profiles
of two closely related New World arenaviruses, the apathogenic Tacaribe virus (TCRV) and the hemorrhagic fever-causing
Junin virus (JUNV), in primary human monocytes and macrophages. Both viruses grew robustly in VeroE6 cells; however,
TCRV titres were decreased by approximately 10 fold compared to JUNV in both monocytes and macrophages. Infection of
both monocytes and macrophages with TCRV also resulted in the release of high levels of IL-6, IL-10 and TNF-a, while levels
of IFN-a, IFN-b and IL-12 were not affected. However, we could show that the presence of these cytokines had no direct
effect on growth of either TCRV of JUNV in macrophages. Further analysis also showed that while the production of IL-6 and
IL-10 are dependent on viral replication, production of TNF-a also occurs after exposure to UV-inactivated TCRV particles
and is thus independent of productive virus infection. Surprisingly, JUNV infection did not have an effect on any of the
cytokines examined indicating that, in contrast to other viral hemorrhagic fever viruses, macrophage-derived cytokine
production is unlikely to play an active role in contributing to the cytokine dysregulation observed in JUNV infected
patients. Rather, these results suggest that an early, controlled immune response by infected macrophages may be critical
for the successful control of infection of apathogenic viruses and prevention of subsequent disease, including systemic
Citation: Groseth A, Hoenen T, Weber M, Wolff S, Herwig A, et al. (2011) Tacaribe Virus but Not Junin Virus Infection Induces Cytokine Release from Primary
Human Monocytes and Macrophages. PLoS Negl Trop Dis 5(5): e1137. doi:10.1371/journal.pntd.0001137
Editor: Thomas William Geisbert, University of Texas Medical Branch at Galveston, United States of America
Received October 18, 2010; Accepted February 16, 2011; Published May 10, 2011
Copyright: ? 2011 Groseth 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 in part by fellowships from the Canadian Institutes of Health Research (AG; www.cihr-irsc.gc.ca), the Schering Foundation (TH;
www.scheringstiftung.de) and the Deutsche Forschungsgemeinschaft (SFB 593, SB; www.dfg.de), as well as a scholarship from the Ju ¨rgen Manchot Foundation
(SW; www.manchot.org) and funding from the Philipps University Marburg (www.uni-marburg.de). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
The Arenaviridae constitute a large family of bi-segmented single-
stranded RNA viruses, with 25 species currently being recognized
[1,2,3,4] and new members being discovered approximately once
every 3 years . The arenaviruses include several significant
human pathogens capable of causing hemorrhagic fever (HF), and
as such they represent a significant threat to human health
worldwide. In particular, members of the New World arenavirus
serocomplex are responsible for at least 5 distinct HF diseases,
collectively termed the South American hemorrhagic fevers, which
are associated with a combination of hemorrhagic and neurologic
symptoms [2,6]. Of these, the most clinically significant in terms of
disease burden is Junin virus (JUNV), which is the causative agent
of Argentine hemorrhagic fever (AHF). Since it was first identified
in the 1950s JUNV infection has typically been responsible for
300–1000 cases of AHF per year. However, since the early 1990s
case numbers of AHF have dropped dramatically due to an
effective targeted vaccination program in the endemic region
[7,8]. Infection is most commonly seen among agricultural
workers and occurs seasonally in the humid pampas of the
provinces of Buenos Aires, Santa Fe, Co ´rdoba, and La Pampa in
Argentina, with infection believed to occur through cutaneous or
mucosal contact to contaminated blood, feces or urine from
infected rodents, often in the form of aerosols . The majority of
these infections with JUNV result in clinical disease , which can
be divided into three distinct phases: prodromal, neurological–
hemorrhagic, and convalescent . The prodromal phase occurs
during the first week after onset of symptoms and can include non-
specific symptoms such as fever (38–39uC), malaise and myalgia as
well as petechiae on the soft palate and cutaneous petechiae .
Subsequently, 20–30% of AHF cases progress to the neurologic–
hemorrhagic phase of disease characterized by severe hemorrhagic
or neurological manifestations and shock .
Despite being responsible for a growing number of hemorrhagic
fevers, the pathophysiological mechanisms underlying the devel-
opment of disease, and particularly hemorrhagic disease, during
arenavirus infection are poorly understood. However, the
www.plosntds.org1May 2011 | Volume 5 | Issue 5 | e1137
involvement of elevated cytokines levels in the pathophysiology of
AHF is suggested by descriptive studies of infected patients where
elevated levels of interferon (IFN)-a, interleukin (IL)-6, IL-8, IL-10
and tumour necrosis factor (TNF)-a have been reported
[10,11,12]. Further, the levels of these cytokines have been shown
to correlate with disease severity, strongly suggesting a role in
pathogenesis [10,11,12]. Thus it seems possible that some of the
processes related to the development of hemorrhagic fever in
JUNV patients may parallel those known to occur in better
characterized viral hemorrhagic fevers, such as those caused by
filoviruses. However, despite evidence for a role in the patho-
physiology of Argentine hemorrhagic fever, data regarding the
source of the cytokines detected in patients during infection is
presently lacking. As with many other hemorrhagic fever viruses,
infection of macrophages as a primary target cell type is believed
to play a crucial role in the establishment of arenavirus infections
[13,14,15], making them a promising candidate as the source of
these elevated cytokine levels. Indeed, for filoviruses it has been
shown that this infection triggers the secretion of high levels of pro-
inflammatory cytokines, particularly TNF-a [16,17], which then
cause changes in the permeability of the vascular endothelium that
lead to the clinical manifestations of hemorrhage and shock .
In addition to New World arenaviruses causing South American
hemorrhagic fever, a number of human apathogenic New World
arenaviruses have also been identified. Interestingly, in some cases
these viruses are closely related to HF-causing arenaviruses. For
instance, Tacaribe virus (TCRV) is closely related to JUNV,
however, despite over 60 years of research on this virus only a
single case of febrile disease has ever been reported [18,19,20].
The close phylogenetic relationships between such pairs of virulent
and avirulent family members provide an excellent opportunity to
identify critical differences in pathogenesis related processes. Using
this approach, we have compared the effects of TCRV and JUNV
infection on human monocytes and macrophages, with a
particular emphasis on the production of cytokines known to be
associated with AHF infection, in order to determine what role the
production of macrophage-derived cytokines in response to virus
infection might be playing in arenavirus pathogenesis.
We found that, despite showing robust growth in VeroE6 cells,
TCRV replication is reduced in primary human monocytes and
macrophages, in comparison to JUNV, and is accompanied by
significant up-regulation of IL-6, IL-10 and TNF-a. However,
these cytokines do not appear to be directly responsible for the
diminished virus titres in these cells. Further, IL-6 and IL-10
production were shown to be dependent on productive infection,
while TNF-a production was not. In contrast to TCRV, JUNV
infection did not stimulate production of any of the cytokines
examined. Thus these observations support a model of arenavirus
pathogenesis in which early cytokine production as a result of
macrophage infection may actually be protective.
Materials and Methods
Viruses and continuous cell lines
Tacaribe virus and Junin virus (strain Romero) were kindly
provided by Dan Kolakofsky (University of Geneva) and Heinz
Feldmann (Public Health Agency of Canada), respectively, and
stocks were prepared as previously described . Vesicular
stomatis virus (VSV) was provided by Friedemann Weber (Philipps
Universita ¨t Marburg) and Sendai virus (SeV) was provided by
Marianne Nain (Philipps Universita ¨t Marburg). All work with
JUNV was carried out in the BSL-4 laboratory at the Philipps
Universita ¨t Marburg.
VeroE6 (African green monkey kidney) cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen)
supplemented with 10% fetal bovine serum (FBS, PAN Biotech),
2 mM L-glutamine (Invitrogen), 100 U/ml penicillin and 100 mg/
ml streptomycin (Invitrogen) and grown at 37uC and 5% CO2.
Isolation and culture of primary human monocytes and
Leukocyte-enriched buffy coats from healthy anonymous
donors were obtained from the blood bank of the Marburg-
Gießen University Hospital. Samples were layered on 15 ml of
Lymphocyte Separation Medium (1.77 g/ml; PAA Laboratories)
in a 50 ml tube. The blood was centrifuged at 6006g for 30 min
at room temperature (RT) with no brake. The layer formed by the
mononuclear cells was carefully removed and washed twice with
50 ml of ice-cold PBS supplemented with magnesium and calcium
(+Mg/+Ca). After each wash step the cells were spun down at
3006g for 7 min at 4uC. After the second centrifugation, the cells
were resuspended in RPMI-1640 with 2% human AB serum
(Sigma) at a concentration of 1.56106cells/ml, and 4 ml per well
was seeded into Primaria 6-well plates (BD Biosciences) and
incubated at 37uC and 5% CO2. After one hour, the medium was
removed and the cells were thoroughly washed three times with
PBS (+Mg/+Ca) to remove the lymphocytes. Then 4 ml of RPMI-
1640 with 5% human AB serum was added to the cells, and they
were incubated for 1 day (monocytes) or 7 days (macrophages) at
37uC and 5% CO2before use.
Virus infection of VeroE6 cells
VeroE6 cell monolayers with a confluence of 80–90% were
infected in 6-well plates with TCRV or JUNV at an MOI of 1 or
0.1 in 1 ml of serum-free DMEM for 60 min at 37uC in a 5%
CO2 atmosphere. Following absorption the inoculum was
removed and the cells washed 3x with DMEM to remove any
unbound virus. Cells were placed in fresh DMEM containing 2%
FBS (PAN Biotech), 2 mM L-glutamine (Invitrogen), 100 U/ml
penicillin and 100 mg/ml streptomycin (Invitrogen) and incubated
for 5 days. Samples from the supernatant were collected every
24 h for analysis of progeny virus release by plaque assay.
It remains unclear how arenavirus infection causes disease;
however, for other hemorrhagic fever viruses, infection has
been linked to over-production of numerous cytokines by
macrophages that can then affect vascular integrity. In
order to determine if a similar mechanism might
contribute to arenavirus pathogenesis, we have examined
the infection and subsequent cytokine production in
human monocytes and macrophages by two closely
related arenaviruses: the apathogenic Tacaribe virus (TCRV)
and the hemorrhagic fever-causing Junin virus (JUNV). We
found that both viruses infected primary monocyte and
macrophage cultures; however, only, in the case of TCRV
was infection accompanied by the production of cyto-
kines. These cytokines would have the potential to
stimulate an antiviral response to infection, including the
production of antibodies, which are known to be
protective during infection. Surprisingly, in contrast to
what is observed in other viral hemorrhagic fevers, we
found that JUNV infection did not have any effect on the
expression of these cytokines. This suggests that an early,
strong immune response by infected macrophages may be
critical for the control of infection by apathogenic
arenaviruses and the prevention of subsequent disease.
Cytokine Expression during Arenavirus Infection
www.plosntds.org2May 2011 | Volume 5 | Issue 5 | e1137
Virus infection of primary human monocytes and
Monocyte or macrophage cultures generated as described above
were infected in 6-well Primaria (BD Biosciences) plates with
TCRV or JUNV at an MOI of 0.1 or mock infected, in 1 ml of
serum-free RPMI-1640 for 60 min at 37uC in a 5% CO2
atmosphere. Following absorption the inoculum was removed
and the cells washed 3x with RPMI-1640 to remove any unbound
virus. Cells were placed in fresh RPMI-1640 containing 2%
human AB serum (Sigma), 2 mM L-glutamine (Invitrogen),
100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen)
and incubated for 4 days. Samples were collected daily for analysis
of progeny virus release by plaque assay and after 4 days for
cytokine release. As positive controls for IFN-a, IFN- b and IL-12
production, monocyte and macrophage cultures were infected
with 64 or 640 HAU of SeV per well, or stimulated with
lipopolysaccharide (LPS, Sigma) at a concentration of 100 ng/mL
or 1 mg/ml. All samples infected with SeV or stimulated with LPS
were harvested after 20 h for analysis of cytokine release.
Plaque assay for titre determination
VeroE6 cell monolayers with a confluence of 80–90% were
infected in a 6- or 12-well plate format with ten-fold dilution series of
samples containing TCRV or JUNV for 60 min at 37uC in a 5%
CO2 atmosphere in serum-free DMEM. For 12-well plates an
infection volume of 250 ml was used, while for 6-well plates an
infection volume of 500 ml was used. Plates were rocked every 15
minutes during infection. Following absorption the inoculum was
removed and the monolayers were then overlaid with 2 ml (12 well)
or 4 ml (6 well) of Minimum Essential Medium (MEM) containing
BactoAgar (BD Biosciences) at a final concentration of either 0.7%
(Invitrogen), 100 U/ml penicillin and 100 mg/ml streptomycin
(Invitrogen). Plaques were allowed to develop for either 5–6 days
(JUNV) or 7–8 days (TCRV) before being fixed and stained with a
0.1% crystal violet solution (10% formaldehyde, 0.1% crystal violet).
The presence of IFN-a and IFN-b (Pestka Biomedical
Laboratories), as well as IL-6, IL-10, IL-12 and TNF-a (R&D
Systems) were detected using commercial assays according to the
manufacturer’s directions. Monocyte and macrophage superna-
tants were analysed undiluted Due to the high signals obtained,
analysis of TCRV samples for IL-6, IL-10 and TNF-a was also
carried out using samples diluted 1:10 in the sample dilution
buffers provided by the manufacturer.
from infected monocytes and macrophages, these supernatants, or
known concentrations of recombinant human IFN-b (Calbiochem)
were incubated on 80% confluent VeroE6 cells for 18 h. Cells were
then washed once with DMEM and infected with vesicular stomatitis
virus (VSV) at an MOI of 0.001 for 1 h at 37uC. Following infection
the inoculum was removed and fresh DMEM containing 2% FBS
(PAN Biotech), 2 mM L-glutamine (Invitrogen), 100 U/ml penicillin
and 100 mg/ml streptomycin (Invitrogen) was added. Cells were then
incubated overnight at 37uC with monolayers examined for the
extent of CPE formation after 12, 18 and 24 h.
Inactivation of arenaviruses by UV irradiation
Stock preparations of TCRV or JUNV with a known starting
titre of 16106pfu/ml (JUNV) or 26106pfu/ml (TCRV) were
inactivated as previously described [22,23] by irradiation at
254 nm using a UV Lamp (CAMAG) for 1 h. Samples of virus
stocks inactivated using this method were analyzed by plaque assay
as described above, to ensure complete inactivation (data not
Removal of virus from cell culture supernatants by
To generate virus-free cytokine-containing supernatants, 1 ml
aliquots of supernatant from TCRV, JUNV or Mock infected
monocytes were loaded into 1.5 ml polyallomer microfuge tubes
(Beckmann) and centrifuged at 55,000 rpm in a TLA-55 rotor
(186,0006g; Beckmann) for 2.5 h at 4uC. Subsequently 900 ul
from each aliquot was transferred to a fresh tube and centrifuged
again under the same conditions. Finally 800 ul was transferred to
a fresh tube. Clarified supernatants were examined by plaque
assay to confirm the removal of all infectious virus (data not
Treatment of macrophages with monocyte culture
Cytokine-containing supernatants, derived from infected mono-
cyte cultures from which virus had been removed, were applied to
new macrophages cultures prepared in 6 well plates as described
above. Cells were then incubated at 37uC with the relevant
supernatant for 2 h prior to infection. Stock virus with a known
titre was then added to the supernatant to generate an MOI of 0.1
per cell. Cells were then incubated for an additional 1 h at 37uC.
Following absorption the inoculum was removed and fresh RPMI-
1640 containing 2% human AB serum (Sigma), 2 mM L-
glutamine (Invitrogen), 100 U/ml penicillin and 100 mg/ml
streptomycin (Invitrogen) was added. Supernatants were harvested
after 4 days for analysis of virus growth.
Junin and Tacaribe replicate with comparable efficiency
in VeroE6 cells, but not in primary monocytes and
Before beginning our analyses in macrophages, we first
compared the replication of TCRV and JUNV by examining
the kinetics of virus growth for these two viruses in VeroE6 cells,
which are known to be broadly permissive for arenavirus infection
. Cells were infected at MOIs of 1 or 0.1. However, regardless
of the MOI used to establish the infection, no significant difference
was observed between TCRV and JUNV during growth in
VeroE6 cells (Fig. 1A). Further, neither virus produced a marked
cytopathic effect (CPE) during the first 4 days of infection, during
which time logarithmic virus growth occurred. However, at later
time points TCRV produced a strong CPE and ultimately resulted
in almost complete detachment of the monolayer (Fig. 1B). In
contrast, for JUNV only limited cell rounding and in some cases
the formation of small transient holes in the monolayer was
observed at similar time points. These data indicate that under
permissive conditions no fundamental defect exists that compro-
mises the capacity of TCRV for replication, in comparison to
In contrast, infection of primary human monocytes and
macrophages with JUNV at an MOI of 0.1 resulted in significantly
faster growth and approximately 10-fold higher end-point titres
compared to TCRV infection (Fig. 2A and 2B). It was also
observed that, while growth of TCRV was reduced compared to
JUNV in both cell types, differences during growth in macro-
Cytokine Expression during Arenavirus Infection
www.plosntds.org3May 2011 | Volume 5 | Issue 5 | e1137
phages were only present at later time points (days 3 and 4), while
differences in monocytes were observed at all time points.
Interestingly, while neither virus produced significant CPE during
infection of macrophages (data not shown), a characteristic CPE
was observed in monocytes during TCRV infection. In these
samples we observed the formation of cell clusters throughout the
culture (Fig. 2C), which resembled those reported for filovirus
infected monocyte cultures , and was suggestive of activation.
TCRV infection of primary monocytes and macrophages
leads to production of IL-6, IL-10 and TNF-a
In order to clarify a potential role for macrophage-derived
cytokines in the pathogenesis of New world arenavirus infections,
we next examined the production of several key cytokines (IFN-a,
IFN-b, TNFa, IL-6, IL-10 and IL-12) following infection of
primary human monocyte and macrophage cultures with either
TCRV or JUNV at an MOI of 0.1. Supernatants were harvested
at day 4 post-infection and examined using commercial cytokine
ELISAs. This time-point was consistent with the accumulation of
maximum virus titres in the cultures and the end of productive
virus growth for both viruses (Fig. 2A and 2B). Surprisingly, we
observed that JUNV infection did not up-regulate any of the
cytokines examined (Fig. 3). However, TCRV infection led to the
release of significant amounts of TNFa, as well as IL-6 and IL-10
(Fig. 3). These levels constituted an up-regulation in comparison to
both JUNV infected and mock infected cells. IL-12 production
was not affected by JUNV or TCRV infection and, surprisingly,
neither IFN-a nor IFN-b were up-regulated in either the
monocyte or macrophage cultures (Fig. 3). This was despite the
observation that both monocytes and macrophages prepared in
this fashion were able to produce detectable levels of IFN-a and
IFN-b in response to SeV infection, and IL-12 in response to LPS
stimulation (data not shown). In order to confirm the absence of
IFN-a and IFN-b in JUNV and TCRV infected supernatants, we
further analysed them using an IFN bioassay. This assay is based
on the ability of IFN-a and IFN-b to inhibit VSV infection of
various cell types, which can then be clearly seen as a reduction in
CPE formation and monolayer loss [25,26]. Using this approach
we could confirm that supernatants derived from JUNV, TCRV
and mock-infected monocyte cultures did not contain significant
amounts of IFN-a/b (Fig. 4). Further, based on comparison with
standards containing known amounts of recombinant human IFN-
b we could determine that all supernatant contained less than the
equivalent of 100 pg/ml (27 IU) of IFN-b, which approached the
background level of detection in the commercial ELISA assays
used, thus confirming the absence of IFN in these samples.
Infection is required for the production of cytokines in
response to Tacaribe virus
In order to determine whether cytokine production by TCRV
infected primary human monocytes and macrophages is depen-
dent on productive infection of these cells, we next exposed fresh
monocyte cultures to UV-inactivated TCRV or JUNV and
compared cytokine production to that in cells infected with
untreated virus. Monocyte cultures were chosen for this analysis as
they showed the highest levels of cytokine production and,
therefore, provided the most sensitive system for comparison of
the cytokine response before and after virus inactivation. We first
confirmed that UV-inactivation resulted in reduction of virus titres
to less than 1 pfu/ml by analysing samples of inactivated virus
stocks via plaque assay (data not shown), consistent with previous
reports demonstrating the efficacy of this method [22,23].
Supernatants from monocytes cultures infected with these
inactivated virus samples were collected after 4 days and examined
for production of TNFa, IL-6 and IL-10 by ELISA. UV-
inactivated JUNV particles were unable to stimulate production
of TNFa, IL-6 or IL-10 (data not shown). When cells were treated
with UV-inactivated TCRV almost no release of IL-6 or IL-10
was detected, in contrast to infection with infectious TCRV (Fig. 5).
Interestingly, when UV-inactivated TCRV was used TNF-a levels
were not significantly reduced (Fig. 5). These observations then
clearly indicate that the production of IL-6 and IL-10 during
TCRV infection of monocytes is strongly dependent on productive
virus infection, while TNF-a production is largely independent of
viral replication. Further, this result demonstrates that IL-6 is not
being induced as a by-product of TNF-a up-regulation in the
monocyte cultures, but is separately and independently regulated
by TCRV infection.
Figure 1. Comparison of virus growth and CPE formation
during TCRV and JUNV infection of VeroE6 cells. (A) Virus growth
during TCRV and JUNV infection. Sub-confluent VeroE6 cells were
infected in a 6 well format with either TCRV or JUNV at an MOI of either
0.1 (upper panel) or 1 (lower panel). Cell culture supernatants were
harvest immediately after infection and every 24 h thereafter for a
period of 5 days. Virus titres in these supernatants were determined by
plaque assay (B) CPE formation during TCRV and JUNV infection. VeroE6
cells were infected as described for (A) and CPE formation was
monitored. Data are shown starting from the time of onset of CPE
Cytokine Expression during Arenavirus Infection
www.plosntds.org4May 2011 | Volume 5 | Issue 5 | e1137
Cytokine production in response to Tacaribe virus
infection does not directly impair arenavirus infection in
Since TCRV infection in both monocytes and macrophages is
somewhat impaired compared to infection with JUNV, we were
interested to determine whether there is a direct effect of the
cytokines produced during TCRV infection on arenavirus
infection in these cell types. To assess this, we first removed virus
particles from the TCRV culture supernatants by ultra-centrifu-
gation. The complete removal of TCRV (residual titres ,1 pfu/
ml) using this method was verified by plaque assay (data not
shown). These clarified supernatants were then used to pre-treat
fresh macrophage cultures before JUNV or TCRV, corresponding
to an MOI of 0.1, was added. Macrophage, rather than monocyte
cultures were used because they generated higher virus titres
during infection, and thus provided a more sensitive system to
analyse any effect on virus growth. These experiments revealed no
influence of the cytokines contained in TCRV-infected monocyte
supernatants on the titres obtained following either TCRV or
JUNV infection (Fig. 6). Thus the production of TNF-a, IL-6 and
IL-10 during TCRV infection does not seem to directly influence
virus replication in these cells.
Taken together, our data clearly demonstrate that, while
pathogenic New World arenavirus infection is not associated with
the production of cytokines in primary human monocytes or
macrophages, infection with the non-pathogenic TCRV produced
significant levels of IL-6, IL-10 and TNF-a. The production of
these cytokines was further shown to be mechanistically distinct as
both IL-6 and IL-10 production were dependent on productive
virus infection while TNF-a production was also induced by UV-
inactivated particles. Further, we found that TCRV has a reduced
ability to replicate in these important primary target cells,
although we could show that this was not directly attributable to
the presence of the cytokines produced in response to infection.
The observation that the infection of macrophages with many
different VHFs consistently induces an inflammatory cytokine
response  has led to a general model for VHF pathogenesis in
which excessive macrophage activation leads to induction of a
cytokine storm. This systemic expression of elevated levels of
cytokines is then proposed to result in the development of a septic
shock-like syndrome, characterized by hypotension, insufficient
tissue perfusion and multiple organ dysfunction , a disease
profile that is consistent with what is known of fatal cases from
many of the hemorrhagic fevers, including those caused by
arenaviruses. While it has been shown that in vitro replication of
LASV in macrophages occurs without inducing production of
inflammatory cytokines [29,30], the lack of coagulation disorders
, as well as the low incidence of severe disease and
corresponding low case fatality rates (1–2%), raise questions as
to whether LASV can be considered as a representative VHF. In
contrast, the New World Arenaviruses produce much more typical
hemorrhagic fevers, characterized by severe disease with high case
fatality rates (15–30%) and coagulation abnormalities. On this
Figure 2. Comparison of virus titre and CPE production during infection of primary human monocytes and macrophages. Growth
kinetics of TCRV and JUNV in (A) primary human monocytes and (B) primary human macrophages. Primary human monocytes and macrophages
were isolated by adherence of peripheral blood mononuclear cell fractions in 6-well plates with subsequent maturation for either 1 (monocyte) or 7
(macrophage) days. Subsequently, monocyte (upper panel) and macrophage (lower panel) populations were infected at an MOI of 0.1 with either
TCRV or JUNV. Cell culture supernatants were harvest immediately after infection and every 24 h thereafter for a period of 5 days. Virus titres in these
supernatants were determined by plaque assay. (C) Cytopathic effect during infection of primary human monocytes with TCRV and JUNV. The cells
from which supernatants were collected for analysis in (A) were analysed by light microscopy for the formation of cytopathic effect in comparison to
mock infected cells 4 days after infection.
Cytokine Expression during Arenavirus Infection
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basis we considered that infection with these viruses may,
therefore, provide a more suitable model for understanding
arenavirus-induced hemorrhagic fever pathogenesis.
A significant role for macrophages in the pathophysiology of
JUNV infection has been indicated by studies in animal models,
where they have been shown to produce high titres of infectious
virus [31,32]. Further, macrophage maturity has been shown to be
important for age-related resistance to JUNV infection in the rat
model . A role for macrophages in human JUNV infection is
also suggested by observations of productive infection of patient
peripheral blood mononuclear cells (PBMCs) during the acute
phase of disease , as well as the increased levels of several
cytokines (IFN-a, IL-6, IL-8, IL-10 and TNF-a) in patient serum
that are known to be produced by, among others, macrophages
[10,11,12]. However, a definitive link between JUNV infection of
macrophages and cytokine production, as well as an understand-
ing of the role these cytokines play in the development of disease is
notably lacking. In order to address this topic we have examined
the patterns of cytokine expression during infection of primary
monocytes and macrophages with either JUNV or the closely
related TCRV, which is apathogenic for humans. It could be
shown that both viruses grow readily in these cells in vitro, although
TCRV growth was decreased by ,1 log compared to JUNV titres
in both monocytes and macrophages. Since TCRV growth in a
broadly permissive cell line (VeroE6) was comparable we suggest
that this growth impairment is a feature of infection in monocytic
cells, rather than being indicative of a general growth defect in
TCRV. This reduction in infectious virus particle production in
monocytic cells could play a potentially important role in limiting
spread from these cells into additional target tissues. Such a role in
dissemination of virus following their initial infection has been
proposed to be an important function of infected macrophages in
the context of filovirus infection , and is supported by kinetic
analyses of filovirus infected non-human primates . Further,
we observed that, while the growth of TCRV is decreased in
comparison to JUNV in both macrophages and monocytes, this
difference is most prominent at early time points in monocytes,
while in macrophages differences are only apparent at late time
points. This suggests that the basis for inhibition of TCRV growth
in these two cell types may be quite different and that cell
attachment plays a direct role in the ability of monocytic cells to be
infected by TCRV. Further, the early suppression of TCRV
Figure 3. Cytokine production during TCRV and JUNV infection of primary human monocytes and macrophages. (A) Cytokine
production during infection of primary human monocytes. Primary human monocytes were infected at an MOI of 0.1 with either TCRV or JUNV or
mock infected in a 6-well plate with supernatants collected for cytokine analysis. Supernatants were analysed after 4 days using commercially
available cytokine ELISA kits for IFN-a and IFN-b (Pestka Biomedical Laboratories) as well as IL-6, IL-10, IL-12 and TNF-a (R&D Systems) according to the
manufacturer’s instructions. The cytokine concentration for each of three donors (black box), as well as the mean for each group (black bar), is
indicated for each treatment group: TCRV infected (T), JUNV infected (J) or Mock (M). (B) Cytokine production during infection of primary human
macrophages. Primary human macrophages were infected and cytokine analysis performed on collected supernatants as described in (A).
Cytokine Expression during Arenavirus Infection
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growth in monocytes speaks for the involvement of an early step in
the infectious process. It is, therefore, tempting to speculate that
monocytic cell attachment may affect the expression of the as yet
unknown TCRV receptor and thereby influence the susceptibility
of adherent versus non-adherent monocytic cells to TCRV
infection. If this is indeed the case, similar effects on the in vivo
susceptibility of circulating blood monocytes during infection
could then affect the potential for subsequent systemic spread of
the virus and thereby contribute to pathogenesis.
Surprisingly, despite the fact that several pro- and anti-
inflammatory cytokines have been shown to be elevated in
patients with severe and/or fatal JUNV infection [10,11,12], we
could not identify any production of IFN-a, IFN-b, TNF-a, IL-6,
IL-10 or IL-12 during in vitro infection of human monocytes or
macrophages with JUNV. In contrast, infection with the human
apathogenic TCRV resulted in a strong up-regulation of several of
these cytokines (IL-6, TNF-a and IL-10) in a manner that, for IL-6
and IL-10, was dependent on productive virus infection.
Interestingly, for TNF-aactivation was largely independent of
productive infection, a finding that also indicates that induction of
IL-6 and IL-10 are not being induced as a by-product of TNF-a
up-regulation, but rather that these mediators are independently
induced. Although the mechanism by which TNF-a induction
occurs remains elusive, a similar phenomenon has been reported
for a diverse range of viruses, including Ebola virus, Marburg
virus, Herpes Simplex virus and Reovirus [17,37,38]. For Ebola
virus, infection-independent TNF-a activation has been suggested
to occur in response to receptor cross-linking by the viral surface
glycoproteins  and, based on our observations, we can propose
that a similar process may take place with arenavirus particles as
well. Considering the similar morphologies demonstrated by the
different arenaviruses, the lack of TNF-a activation with JUNV
UV-inactivated particles might be related of the differing receptor
usage between TCRV and JUNV .
While our findings initially appear to be in contrast with the
established cytokine storm model of viral hemorrhagic fever
pathogenesis, as well as JUNV patient data, which clearly shows
up-regulation of numerous cytokines, we believe that these
findings can be reconciled. In filovirus patients it was observed
that a strong but transient release of cytokines early in infection is
linked to patient survival [41,42], while terminal patients develop
some of the same cytokines but only late in infection . Thus, it
appears not to be the production of proinflammatory cytokines as
such that is devastating for the infected patient, but rather their
uncoordinated release. In further support of such a model it has
been shown that macrophages infected in vitro with LASV also
support virus replication, but without activation or induction of
cytokine responses, while infection with the closely related but
apparently human apathogenic Mopeia virus results in up-
regulation of mRNAs encoding IFN-a, IFN-b, TNF a and IL-6
[29,30,43]. Taken together with our data, this seems to suggest
that strong cytokine activation during macrophage infection is a
feature of apathogenic arenavirus infection, regardless of whether
the virus in question is an Old World or New World arenavirus.
Further, an ability to suppress cytokine activation during
macrophage infection may be equally typical of pathogenic family
members. Thus our findings seem to be in agreement with an
increasing body of evidence suggesting that an early induction of
cytokines and the corresponding innate immune responses can
play a protective role in preventing the development of
hemorrhagic fever during arenavirus infection.
The absence of IFN-a during JUNV infection of monocytes and
macrophages was particularly unexpected, given the very high
levels of IFN-a known to be present in infected patients. This
finding clearly suggests that the IFN-a found during infection is of
non-monocytic origin and is rather derived from other leukocyte
populations. In particular, the potential of dendritic cell popula-
tions as sources of interferon during infection will have to be
closely examined. The lack of IFN production during JUNV
infection can also potentially be explained by the existence of two
known viral IFN antagonists, NP and Z [44,45]. However, the
absence of appreciable IFN-a or IFN-b production during TCRV
infection was surprising. This finding suggests that, at least in
monocytic cells, TCRV is capable of circumventing the type-I
interferon response. This is despite evidence indicating that
TCRV NP is impaired in its ability to inhibit IFN induction,
compared to other arenaviruses NPs . While JUNV Z has
recently been suggested to serve as an additional interferon
antagonist for JUNV , there is currently no information
regarding a similar function for TCRV Z. Thus, it remains
possible that this or other viral proteins may have as yet
undescribed interferon antagonistic activities. Alternatively, it has
also been suggested that TCRV may be able to avoid triggering
the interferon response during infection by means of an additional
non-coded terminal nucleotide in the virus genome, which is
added during replication  and may mask the 59 tri-phosphate
Figure 4. Bioassay for IFN production during TCRV and JUNV
infection of primary human monocytes. IFN-a/b activity in
monocyte culture supernatants was detected using a biological assay
for protection against vesicular stomatitis virus (VSV) infection and its
associated cytopathic effects. For this assay, VeroE6 cells cultured in 12-
well plates were treated with supernatants from monocytes infected
with either JUNV or TCRV, supernatants from mock infected monocytes
or dilutions of a commercial recombinant human IFN-b standard
(50 ng/ml to 100 pg/ml). After 24 h the cells were washed and infected
with VSV at an MOI of 0.001. Cells were then incubated at 37uC for 18 h
before being examined for the formation of CPE.
Cytokine Expression during Arenavirus Infection
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RNA end that serves as a trigger for activation of the interferon
cascade through RIG-I . Together with previous reports
showing that the nucleoprotein of Pichinde virus, another non-
human pathogenic New World arenavirus, acts as an efficient
interferon antagonist , our own findings support the position
that evasion of the interferon response is necessary for virus
survival in both pathogenic and apathogenic viruses, rather than
being an inherent indicator of pathogenic potential.
One possibility that was examined in this study was that the
high levels of cytokine production in monocytes and macrophages
during TCRV infection might play a direct role in restricting virus
growth in these cells. However, an analysis of the growth of both
JUNV and TCRV in macrophages pre-treated with TCRV-
derived cytokine-containing supernatant showed no influence of
these cytokines on the virus titres obtained following infection.
This observation indicates that any protective role of these
cytokines is unlikely to be due to a direct role in limiting TCRV
replication in macrophages. However, they may still play a
systemic role in regulation of the immune response to TCRV
infection. In light of the mounting evidence showing a role of
macrophage-derived cytokine expression in apathogenic arenavi-
rus infection, we propose that up-regulation of IL-6 and TNF-a in
the context of TCRV infection appropriately activates the acute
phase response rather than contributing to immunopathology. In
contrast, the absence of IL-6 and TNF-a production after infection
with JUNV might allow the virus to continue to replicate
unchecked and thereby achieve the high virus loads and broad
tissue dissemination that are indicative of many viral hemorrhagic
fevers . The up-regulation of IL-10 by TCRV is also intriguing
since this cytokine is known to play a role in the activation and
maturation of B-cells. Given the role for the antibody response in
the control of New World arenavirus infection, as is clearly
demonstrated by the success of passive antibody transfer in the
treatment of AHF patients , its up-regulation may also be of
particular significance for preventing the development of disease
during human TCRV infection, While antibody production, and
particularly the production of neutralizing antibody, does occur
during JUNV infection, we would suggest that the induction of IL-
10 following TCRV infection of monocytes and macrophages may
Figure 5. Cytokine production during exposure of primary human monocytes and macrophages to inactivated TCRV. Samples
containing stock TCRV were inactivated by exposure to UV light for 60 min before being used to infect primary human monocytes at a dilution
equivalent to an MOI of 0.1. Infection with untreated TCRV or mock infection of cells served as controls. Supernatants from the infected cells were
analysed after 4 days using commercially available cytokine ELISA kits for IL-6 (left panel), IL-10 (center panel) and TNF-a (right panel) (R&D Systems)
according to the manufacturer’s instructions. The cytokine concentration for each of three donors (black box), as well as the mean for each group
(black bar) is indicated for each treatment group: Untreated TCRV (Untr.), UV-inactivated TCRV (UV) or Mock (M).
Figure 6. Comparison of virus titre during infection in the
presence or absence of cytokine-containing supernatants.
Cytokine supernatants collected from primary human monocytes
infected at an MOI of 0.1 with either TCRV or JUNV or mock infected
at 4 days post-infection were subjected to ultracentifugation to remove
virus particles. Macrophage cultures were then pretreated with the
clarified supernatant of TCRV, JUNV or mock infected monocytes for 2 h
prior to the addition of known amounts of (A) JUNV or (B) TCRV for
infection. Samples were collected after 4 days and plaque assays
performed to determine virus titres.
Cytokine Expression during Arenavirus Infection
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be critical for inducing a stronger and/or more rapid humoral
immune response to infection, which could aid in rapid virus
clearance and thus prevent the onset of clinical disease.
Taken together, this study shows that, unlike the better
understood filovirus hemorrhagic fevers, pathogenic New World
arenavirus infection is associated with a lack of cytokine up-
regulation during in vitro macrophage infection, despite productive
infection. In contrast, infection with the human apathogenic
TCRV was associated with high levels of IL-6, IL-10 and TNF-a.
In conjunction with the observations from severe clinical AHF
cases, which show high levels of these same cytokines in
symptomatic JUNV patients, this finding suggests a more complex
relationship between cytokine production during infection of
primary target cells (i.e. macrophages) and the subsequent
development of disease, and provides much needed insight into
the immune processes necessary for the successful control of
The authors are grateful to Heinz Feldmann, Dan Kolakofsky, Friedemann
Weber and Marianne Nain for providing the virus strains used in this work.
We are also grateful to Friedemann Weber and Hideki Ebihara for their
critical discussion of the manuscript and to Markus Eickmann, Gotthard
Ludwig and Michael Schmidt for their technical assistance with the BSL-4
Conceived and designed the experiments: AG TH AK SB. Performed the
experiments: AG TH MW SW AH AK. Analyzed the data: AG TH AK
SB. Contributed reagents/materials/analysis tools: AG TH AK SB. Wrote
the paper: AG TH AK SB.
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