ORIGINAL ARTICLE

Caraparu virus induces damage and alterations in antioxidantdefenses in the liver of BALB/c mice after subcutaneous infection

Fernanda Caetano Camini • Letıcia Trindade Almeida • Carolina Silva Bernardes •

Maısa Silva • Maria Lucia Pedrosa • Daniela Caldeira Costa • Wanderson Geraldo de Lima •

Carla do Amaral Pinto • Paulo Cesar Peregrino Ferreira • Jose Carlos de Magalhaes •

Cintia Lopes de Brito Magalhaes

Received: 18 September 2013 / Accepted: 14 May 2014

� Springer-Verlag Wien 2014

Abstract Oxidative stress is a disturbance in the oxidant-

antioxidant balance leading to potential cellular damage.

Most cells can tolerate a mild degree of oxidative stress

because they have a system that counteracts oxidation that

includes antioxidant molecules such as glutathione (GSH)

and superoxide dismutase (SOD). Disruption of the host

antioxidant status has been recognized as an important

contributor to the pathogenesis of many viruses. Caraparu

virus (CARV) is a member of group C of the Bunyaviridae

family of viruses. In South American countries, group C

bunyaviruses are among the common agents of human

febrile illness and have caused multiple notable outbreaks

of human disease in recent decades; nevertheless, little is

known about the pathogenic characteristics of these viru-

ses. The purpose of this study was to examine the hepatic

pathogenesis of CARV in mice and the involvement of

oxidative stress and antioxidant defenses on this pathology.

Following subcutaneous infection of BALB/c mice, CARV

was detected in the liver, and histopathology revealed acute

hepatitis. Increased serum levels of aspartate and alanine

aminotransferases (AST/ALT) and greater hepatic expres-

sion of the proinflammatory cytokine tumor necrosis fac-

tor-a (TNF-a) were found in infected animals. CARV

infection did not alter the biomarkers of oxidative stress but

caused an increase in GSH content and altered the

expression and activity of SOD. This is the first report of an

alteration of oxidative homeostasis upon CARV infection,

which may, in part, explain the hepatic pathogenesis of this

virus, as well as the pathogenesis of other Bunyaviridae

members.

Introduction

Oxidative stress is an important contributor to pathogenesis

in many viral diseases, such as hepatitis B, hepatitis C, Rift

Valley fever, respiratory disease caused by respiratory

syncytial virus, and dengue [7, 8, 10, 15–18, 21, 24, 39].

Reactive oxygen species (ROS) are highly unstable mole-

cules that are involved in many forms of tissue damage,

including the damage caused to cellular components such

as lipids, proteins and DNA [14, 15]. However, cells are

protected against ROS and oxidative damage by well-

developed enzymatic and non-enzymatic antioxidant sys-

tems, including superoxide dismutase (SOD), catalase,

glutathione-dependent enzymes, thioredoxin and peroxire-

doxins [17]. Within cells, the redox potential is determined

primarily by the total content of glutathione (GSH) [26].

GSH is particularly important in the liver, where it serves

as the principal non-protein thiol involved in the cellular

F. C. Camini � M. L. Pedrosa � D. C. Costa �W. G. de Lima � C. L. de Brito Magalhaes

Nucleo de Pesquisas em Ciencias Biologicas, NUPEB,

Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais,

Brazil

F. C. Camini � L. T. Almeida � C. S. Bernardes � M. Silva �M. L. Pedrosa � D. C. Costa � W. G. de Lima �C. L. de Brito Magalhaes (&)

Departamento de Ciencias Biologicas, Universidade Federal de

Ouro Preto, Campus Universitario Morro do Cruzeiro,

Ouro Preto, Minas Gerais 35.400-000, Brazil

e-mail: cintia.magalhaes@gmail.com

C. do Amaral Pinto � P. C. P. Ferreira

Departamento de Microbiologia, Universidade Federal de Minas

Gerais, Belo Horizonte, Minas Gerais, Brazil

J. C. de Magalhaes

Departamento de Quımica, Biotecnologia e Engenharia de

Bioprocessos, Universidade Federal de Sao Joao del-Rei,

Ouro Branco, Minas Gerais, Brazil

123

Arch Virol

DOI 10.1007/s00705-014-2123-2

antioxidant defense mechanism. Additionally, antioxidant

enzymes (AOEs) can either directly decompose ROS (e.g.,

SOD and catalase) or facilitate these antioxidant reactions

(e.g., peroxidase using glutathione as a reducing agent).

The first ROS produced in the reduction pathway of oxy-

gen is the superoxide anion (O2-), which is metabolized to

hydrogen peroxide (H2O2) by enzymes of the SOD family.

Higher eukaryotes have three isoforms of SOD: cytoplas-

mic SOD1 (Cu/Zn-SOD), mitochondrial SOD2 (Mn-SOD)

and extracellular SOD3 (Cu/Zn-SOD) [23]. The glutathi-

one redox cycle is complementary to catalase in converting

H2O2 to water and oxygen [41]. Disruption of the host

antioxidant machinery is associated with many disease

states.

The family Bunyaviridae is composed of more than 350

RNA viruses that are classified into five distinct genera:

Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus and

Tospovirus [32]. Bunyavirus particles are approximately

100 nm in diameter and contain three single-stranded RNA

segments. All bunyaviruses encode four structural proteins:

the viral polymerase (L) on the large (L) segment, the

glycoproteins (Gn and Gc) on the medium (M) segment,

and the N protein on the small (S) segment. Viruses within

the genera Orthobunyavirus and Phlebovirus also encode

non-structural proteins, either on the M segment (termed

NSm) and/or on the S segment (NSs) [12]. With the

exception of the hantaviruses, these viruses are mainly

transmitted to mammals by arthropods and cause mild to

severe disease in humans, including fever, encephalitis and

hemorrhagic fever [32]. In tropical and subtropical areas,

bunyaviruses pose an increasing threat to human health and

are considered to be emerging pathogens [34]. In South

American countries, Oropouche (OROV), Guaroa (GROV)

and group C bunyaviruses are among the common agents

of human febrile illness and have caused multiple notable

outbreaks of human disease in recent decades [2, 11, 13,

37].

The genus Orthobunyavirus is subdivided into multiple

serological groups. Among these are the group C viruses,

which were initially discovered in the Brazilian Amazon

region during the 1950s [6, 9, 33]. A total of 14 distinct

group C viruses have been isolated from humans, mos-

quitoes, wild rodents, marsupials and bats [11, 38]. Cara-

paru virus (CARV) was first isolated in 1956 from a

sentinel monkey (Cebus apella) at the Utinga forest in

Belem, Para State, Brazil. Subsequently, CARV was iso-

lated from the blood of a febrile forest worker and from

arthropods in the same region [9]. In humans, the CARV

causes a self-limited disease lasting 2 to 5 days with

symptoms including fever, headache, myalgia, nausea,

vomiting and weakness [19, 25]. In experimentally infected

mice, CARV induces hepatitis [3]. In humans, there have

been no reports of CARV causing hepatitis; however,

autopsy or biomarkers of liver injury data are unavailable.

Interestingly, for another important bunyavirus in Brazil,

OROV, there have been no reports of clinical liver damage

in patients, but altered liver enzymes have been recorded,

and thus the liver is thought to be an important target organ

for human OROV infections [30].

Importantly, group C viruses of the genus Orthobunya-

virus are associated with febrile illness in humans. Because

of their nonspecific nature, they are usually not reported, or

are misdiagnosed, so their true incidence is unknown [2,

11, 13, 37]. Despite the public-health importance of the

group C viruses, little is known about their pathogenic

characteristics. A previous study demonstrated that intra-

peritoneal inoculation of CARV causes coagulative liver

necrosis in B6C3F1 mice; however, the mechanisms of

CARV-induced hepatic disease remain incompletely

defined [3]. Up to now, there have been no studies using

inoculation of experimental animals by subcutaneous

routes, which would more closely resemble the natural

means of CARV infection. Additionally, a non-lethal

mouse model of disease that more closely resembles the

disease in humans is not available. Thus, the aim of this

study was to characterize, in a non-lethal subcutaneous

model for CARV infection in BALB/c mice, the possible

involvement of oxidative homeostasis on hepatic disease.

Following subcutaneous inoculation with CARV, the

animals were monitored for 14 days, and they displayed

conspicuous clinical signs two or three days postinfection

(pi). From the ninth day pi, no clinical signs were evident,

and none of the infected mice died. Aspartate and alanine

aminotransferases (AST/ALT) levels and the liver histo-

pathology of infected animals revealed hepatitis on days 3

and 7 pi. An increase in TNF-a mRNA expression was

observed in infected animals on day 3 pi. No involvement

of oxidative stress was observed in CARV-induced hepatic

injury, because the levels of oxidative stress markers were

similar between infected and control mice. However, the

levels of GSH were increased in infected animals on days

3, 7 and 14 pi. Concomitantly, levels of SOD changed on

different days following exposure to the virus. The SOD1

mRNA expression was downregulated on days 3 and 7 pi,

and SOD2 and SOD3 mRNA expression were upregulated

on day 14 pi. The total SOD activity showed a decrease on

day 3 pi, returned to control levels on day 7 pi, and

increased on day 14 pi.

Our results demonstrate that CARV infection in BALB/

c mice causes acute hepatitis, with strong positivity for

viral antigen, inflammation and liver damage. In this

pathology, we observed an increase in GSH content and an

early decrease followed by a late increase in SOD, but no

significant oxidative stress. These results suggest that

despite the decrease in SOD levels in the initial days pi,

GSH might prevent CARV-induced oxidative stress during

F. C. Camini et al.

123

liver injury in mice. One of the recurrent themes in recent

studies of many infectious diseases is the modulation of

host responses elicited by exposure to the infection.

Materials and methods

Virus stock preparation

CARV (BeAn3994) was obtained from the American Type

Culture Collection (ATCC) and propagated in Vero cell

culture (African green monkey kidney cell line). After viral

adsorption, the cells were washed twice with phosphate-

buffered saline (PBS) and fed with MEM (Cultilab, Brazil)

supplemented with 1 % fetal bovine serum (FBS). After

90 % of the cells exhibited cytopathogenic effects, the

supernatant was centrifuged at 3,000 g for 10 min at 4 �C and

the viral pools were aliquoted and stored at -80 �C. The

virus titer was 106 PFU/ml, as measured using a methylcel-

lulose plaque assay. Briefly, supernatants of virus-infected

Vero cells were diluted in PBS containing 2 % FBS and

subsequently used to infect confluent Vero cells grown in

six-well plates. After a 1-h incubation at 37 �C, the cells were

overlaid with MEM containing 2 % FBS and 1 % carboxy-

methylcellulose (Sigma-Aldrich, Brazil) and incubated at

37 �C for 5 days. Cells were fixed with 4 % formaldehyde in

PBS, and plaques were revealed with Giemsa stain.

Experimental infection of mice and sample collection

BALB/c mice aged 6 weeks were obtained from the Animal

Facility of Rene Rachou Research Center, Oswaldo Cruz

Foundation (Minas Gerais, Brazil), and were housed in filter-

top micro-isolator cages; they were provided with commercial

mouse feed and water ad libitum. Animal experimentation

was carried out in accordance with the regulations of the Ethics

Committee on Animal Research (CEUA) of Universidade

Federal de Ouro Preto (Minas Gerais, Brazil). Twenty-four

animals were infected via the subcutaneous route with 0.1 ml

of viral suspension containing 105 PFU of CARV, and twenty-

four animals were sham inoculated using the same volume of

control medium. Three groups containing sixteen animals each

(eight infected animals and eight uninfected controls) were

anesthetized with ketamine and xylazine and euthanized by

exsanguination on days 3, 7 and 14 pi. Blood samples were

collected and centrifuged for determination of the AST/ALT

serum biomarkers, and the livers were removed and immedi-

ately stored at -80 �C for subsequent analysis.

Study of hepatic function markers

Serum levels of AST and ALT were measured to determine

hepatic function using Labtest kits # 52 and 53 (Minas

Gerais, Brazil) according to the manufacturer’s

instructions.

Histopathology, immunohistochemistry

and morphometric analysis of inflammatory cells

After collection, the livers were fixed in 10 % buffered

formalin, dehydrated in increasing (70 to 100 %) ethanol

concentrations, cleared in xylene and embedded in paraffin.

The tissue blocks were cut into 4- to 5-lm sections with a

microtome, and the sections were stained with hematoxy-

lin/eosin. For viral antigen detection, 5- to 7-lm thick

tissue sections were deparaffinized and treated using the

heat-induced antigen retrieval technique (Target Retrieval

Solution, S1700, Dako Corporation, USA). Subsequently,

the tissue sections were treated with 3.5 % H2O2 in PBS,

blocked with 20 % normal goat serum in PBS for 30 min at

room temperature and incubated overnight at 4 �C with a

1:2,000 dilution of mouse immune ascitic fluid anti-arbo-

virus group C-I (NIH Research Reagent – Catalog No

G201-701-567, USA) in 2 % bovine serum albumin (BSA,

1870, Inlab, Brazil) in PBS. After incubation with the

primary antibody, the tissue sections were washed three

times with PBS and incubated for 30 min at room tem-

perature with the secondary, biotinylated antibody solution

(DAKO Kit K0675, USA) and then washed with PBS and

treated with the tertiary solution containing peroxidase-

conjugated streptavidin (DAKO Kit K0675, USA) for

30 min at room temperature. The sections were then rinsed

in PBS and incubated with 3,30-diaminobenzidine tetrahy-

drochloride (Sigma, USA) (0.05 %) and hydrogen peroxide

(0.03 %) for 5 min. The sections were then rinsed in PBS

and counterstained with hematoxylin (Reagen, Brazil).

Negative controls were prepared using PBS instead of the

primary antiserum. Morphometric measurements of the

inflammatory cells in liver sections (twenty sections/ani-

mal) were made using a light microscope (Leica DM5000)

and analyzed using Leica Qwin Image Processing and

Analysis Software (Germany).

Virus titration

The livers were macerated in MEM 0 % FBS (Cultilab,

Brazil) and centrifuged at 2,000 g for 3 min at 4 �C. The

supernatant was collected, and the viral titer (PFU/g of

liver) was determined using the methylcellulose plaque

assay in Vero cell culture as described above.

Measurement of the biomarkers of oxidative stress

and total glutathione content in liver homogenates

The level of thiobarbituric acid reactive substances

(TBARS) was estimated using the method described by

Changes in oxidative homeostasis in Caraparu virus infection

123

Buege and Aust [4]. Liver homogenate supernatants were

mixed with trichloroacetic acid (TCA 28 % w/v in 0.25 N

hydrochloric acid), thiobarbituric acid (TBA 1 % in

0.25 M acetic acid) and butylated hydroxytoluene (BHT

125 mM in ethanol); they were then heated for 15 min at

95 �C and then placed in an ice bath. The precipitated

material was removed by centrifugation, and the absor-

bance of the samples at 535 nm was determined. The

TBARS level was calculated using the molar absorption

coefficient of malondialdehyde (MDA). Carbonyl protein

levels were determined according to the method described

by Levine [20]. Each sample was precipitated with 10 %

(w/v) TCA. After centrifugation, the precipitate was treated

with 10 mmol of 2,4-dinitrophenylhydrazine (DNPH) in

2 M hydrochloric acid, incubated in the dark for 30 min

and treated with 10 % TCA. After centrifugation, the

precipitate was washed twice with ethanol/ethyl acetate

(1:1) and dissolved in 6 % sodium dodecyl sulfate (SDS).

The absorbance of the samples at 370 nm was determined.

The results were expressed in nmol of DNPH incorporated/

mg of protein. The content of DNPH incorporated was

calculated using the molar absorption coefficient of DNPH

(22,000 M-1cm-1). Total protein content was determined

according to the method described by Lowry [22] using

bovine serum albumin (BSA) as the standard. The total

glutathione content of liver homogenates was determined

using a Glutathione Assay Kit (CS0260) from Sigma (St.

Louis, MO). This assay uses a kinetic method based on the

reduction of 5,50-dithiobis-(2-nitrobenzoic acid) (DTNB) to

5-thio-2-nitrobenzoic acid (TNB), which can be measured

spectrophotometrically at 412 nm.

Quantitative real-time reverse transcription PCR (q-RT-

PCR)

Total RNA was extracted from liver samples from control

and CARV-infected mice using an SV Total RNA Isolation

Kit (Promega, Brazil) according to the manufacturer’s

instructions. The concentration and purity of the RNA were

estimated spectrophotometrically using the A260/A280

ratio (NanoVue, GE Healthcare, Hertfordshere, UK). The

cDNA was synthesized from 2 lg of total RNA with ran-

dom primers using a High-Capacity cDNA Reverse Tran-

scription Kit (Applied Biosystems, Foster City, CA, USA)

according to the manufacturer’s instructions. The q-RT-

PCR was performed using SYBR Green PCR Master Mix

reagent (Applied Biosystems, Foster City, CA, USA) in a

final reaction volume of 12 lL. The reaction included 2 lL

of cDNA (100 ng) and 0.5 lL of each primer (forward and

reverse, 10 lM). The forward and reverse primers

sequences for TNF-a, SOD1, SOD2 and GAPDH were

obtained from the published nucleotide sequences [31, 40].

The primer sequences for SOD3 (forward primer 50-

TTACCACAAGGGACAGCCAA-30 and reverse primer

50-GTTATGTGAGCGAGCAGCAG-30) were derived

from the Mus musculus genome (National Center for

Biotechnology Information GenBank, accession number

NM_011435.3) and were constructed using the Primer-

BLAST program (http://www.ncbi.nlm.nih.gov/tools/pri

mer-blast/). The reactions were carried out under the fol-

lowing conditions: 50 �C for 2 min and 95 �C for 10 min,

followed by 40 cycles of 95 �C for 15 s and 60 �C for

1 min. Dissociation curves of the amplified product were

analyzed to confirm product specificity. The data obtained

were analyzed using the comparative CT method. Target

gene expression was determined relative to the endogenous

GAPDH gene. All analyses were performed in triplicate.

Biochemical assay for the total SOD activity

The activity of the total SOD was measured using a

Superoxide Dismutase Assay Kit (706002) from Cayman

Chemical Company (MI, USA). Briefly, 100 mg of liver

samples were homogenized in cold 20 mM HEPES, pH

7.2, containing 1 mM EGTA, 210 mM mannitol and

70 mM sucrose. Ten microliters of supernatant was used in

the assay. The reaction was initiated by adding xanthine

oxidase. The plate was incubated on a shaker for 20 min at

room temperature, and the absorbance at 450 nm was

measured using a plate reader (Biotek ELx808).

Statistical analysis

Data were analyzed using GraphPad Prism 3.0 software

and expressed as the mean ± standard deviation of eight

different animals. Student’s t-test at 95 % confidence was

used to determine the level of differences between the

CARV-infected groups and uninfected groups, with *, **

and *** representing significant differences at p \ 0.05,

p \ 0.005 and p \ 0.0005, respectively. The letters a, b,

and c represent differences between the groups of CARV-

infected animals, using one-way ANOVA and Tukey’s

post-test.

Results

Clinical findings and evaluation of hepatic function

markers

Control and CARV-infected BALB/c mice were examined

for clinical abnormalities twice daily for 14 days. All of

the infected mice were susceptible to infection and

developed conspicuous clinical signs within two or three

days pi, manifested by prostration, ruffled fur, shivering,

and a weight loss. This weight loss was more pronounced

F. C. Camini et al.

123

on the third and the fifth days pi (Fig. 1). From the ninth

day pi, no clinical sign was evident, and none of the

infected mice died. The mean AST and ALT levels in the

serum were significantly increased in infected animals

relative to the controls at 3 and 7 days pi but not at

14 days pi (Fig. 2a, b). These results indicate that CARV

infection induced disease with notable involvement of

hepatic injury.

Histopathology, immunohistochemistry

and morphometric analysis of inflammatory cells

To better characterize the hepatic effect of CARV infection

in BALB/c mice, histopathological analysis of liver sam-

ples was conducted, and the results revealed alterations that

were characteristic of infection. On days 3 and 7 pi, the

liver parenchyma of infected animals revealed moderate

and multifocal mononuclear inflammatory infiltrates, pre-

dominantly in the parenchymal and portal space; moderate

hyperemia and a suggestive microvesicular steatosis were

also noted (Fig. 3b, f). The histopathological alterations in

the livers of infected mice were more severe on day 7 pi

(Fig. 3f) than on day 3 pi (Fig. 3b) and were absent on day

14 pi (Fig. 3j), when they were similar to the controls

(Fig. 3i). Using virus titration, CARV was recovered from

the livers of infected animals only on day 3 pi, with a viral

titer of 106 PFU/g in the liver. Despite CARV titers being

undetectable in the livers of infected animals on days 7 and

14 pi, CARV antigen was detected by immunohistochem-

istry in all of the infected animals that were analyzed

(Fig. 3d, h, l). In all of the infected mice, positive staining

for the antigen was observed in the cytoplasm of the cells

in several areas of the hepatic parenchyma, with notable

staining on day 3 pi (Fig. 3d). In control animals, there was

no immunoreactivity to the CARV antigen (Fig. 3c, g, k).

Next, to determine the number of inflammatory cells, liver

sections were stained with hematoxylin/eosin and mor-

phometric analysis was performed. There were signifi-

cantly more inflammatory cells in the livers of infected

mice relative to the controls on days 3 and 7 pi, with the

numbers peaking on day 7 pi (Fig. 4), which confirmed the

histopathological findings.

TNF-a gene transcription is upregulated in infected

mice

It is well documented that multiple factors from bacteria,

viruses and parasites stimulate the production of TNF-a in

the host. To analyze the importance of TNF-a in the

hepatic injuries caused by CARV infection, expression of

the TNF-a gene was evaluated by q-RT-PCR analysis. The

TNF-a mRNA expression peaked in the livers of infected

mice on the third day pi, with approximately a 4.5-fold

increase compared to the control mice (Fig. 5). On days 7

and 14 pi, the TNF-a mRNA expression in infected mice

was 2.3-fold and 2-fold higher, respectively, than in the

Fig. 1 Percentage body weight in BALB/c mice infected with

CARV. The body weight of control and CARV-infected BALB/c

mice was recorded on different days after inoculation. The data are

expressed as percentage of weight and as the mean ± standard

deviation (n = 8 per group). ** and *** indicate significant

differences relative to the control group at p \ 0.005 and

p \ 0.0005, respectively

Fig. 2 Serum levels of aspartate and alanine aminotransferases

(AST/ALT). (a) ALT activity. (b) AST activity. Sera from control

and CARV-infected BALB/c mice were used to determine ALT and

AST activity levels on different days after inoculation. The data are

expressed as the mean ± standard deviation (n = 8 per group). **

and *** indicate significant differences relative to the control group at

p \ 0.005 and p \ 0.0005, respectively. The letters a, b, and c

indicate differences between the groups of CARV-infected animals

Changes in oxidative homeostasis in Caraparu virus infection

123

control groups, but these differences were not significant.

Thus, since it is well documented that TNF-a regulates a

broad range of biological events, including inflammation,

elevated levels of TNF-a in liver of BALB/c mice after

CARV infection could be associated with the higher

inflammation in this tissue.

Fig. 3 Hematoxylin-eosin histopathology and immunohistochemis-

try of the livers of control and CARV-infected mice on days 3, 7 and

14 pi. Inflammatory cells, hyperemic blood vessels and microvesic-

ular steatosis (black arrow in f) were observed in infected animals on

day 3 pi (b) and day 7 pi (f). Note the absence of histological

alterations in control animals (a, e, i) and infected animals (j) on day

14 pi. Cells that were positively immunolabeled for the CARV

antigen were observed on each day (white arrows in d, h, and l). In

control animals, there was no immunoreactivity to the CARV antigen

(Fig. 3c, g, k). Control immunohistochemistry image (inset in c),

without incubation with primary antiserum. Bar, 50 lm (440 9 mag-

nification). SC, sinusoid capillary; CVL, central vein lobular; HP,

hepatic parenchyma; HBV, hyperemic blood vessel; IF, inflammatory

foci

Fig. 4 Morphometric measurements of inflammatory cells in the

livers of BALB/c mice. The numbers of inflammatory cells in liver

sections of control and CARV-infected BALB/c mice were counted

on different days. Twenty sections per animal were counted, and the

data are expressed as the mean ± standard deviation. * and ***

indicate significant differences relative to the control group at

p \ 0.05 and p \ 0.0005, respectively. The letters a, b, and c indicate

differences between the groups of CARV-infected animals

Fig. 5 Alteration in the level of the TNF-a mRNA following

infection by CARV. The total RNAs from livers of control and

CARV-infected BALB/c mice were analyzed by q-RT-PCR with

primers for TNF-a. The results were normalized to GAPDH RNA

levels. The data are expressed as the mean ± standard deviation

(n = 8 per group). ** indicates a significant difference relative to the

control group at p \ 0.005. The letters a and b indicate differences

between the groups of CARV-infected animals

F. C. Camini et al.

123

Effect of CARV infection on the biomarkers

of oxidative stress and total glutathione content

To determine if CARV infection induces oxidative stress

directly in the livers of mice, lipid peroxidation and oxi-

dative modification of proteins were monitored using the

levels of the biomarkers TBARS and carbonyl protein,

respectively. The levels of both markers were not signifi-

cantly different in infected and control groups (Table 1).

Because previous studies have shown that depletion of

intracellular GSH levels is correlated with increased gen-

eration of ROS and the progression of many viral diseases,

we investigated if CARV infection alters the hepatic con-

tent of GSH. As shown in Fig. 6, there was an increase in

the hepatic GSH content in the CARV-infected groups on

all days, with 20, 15 and 40 % increases relative to control

groups on days 3, 7 and 14 after infection, respectively.

Thus, although it is difficult to determine the precise

mechanisms by which GSH provided protection in the

context of CARV infection, maintenance of reductive

intracellular environment is likely a key one.

CARV infection modifies the expression and activity

of the antioxidant enzymes SOD1, SOD2 and SOD3

The first ROS produced in the reduction pathway of oxy-

gen is O2-, which is metabolized to H2O2 by SOD1, SOD2

or SOD3, depending on the primary site of O2- production.

Altered SOD levels have been associated with many

infectious and non-infectious disease states. To investigate

the effect of CARV infection on the expression and activity

of SOD (a protective mechanism against oxidative damage

in the livers of mice), expression of the SOD1, SOD2 and

SOD3 genes and total SOD activity were evaluated by

q-RT-PCR and colorimetric assays, respectively.

Total RNA was isolated from the livers of control and

CARV-infected mice on days 3, 7 and 14 pi, and the

SOD1, SOD2 and SOD3 mRNAs were amplified by

q-RT-PCR. In CARV-infected mice, there was a signifi-

cant decrease in SOD1 mRNA expression on days 3 and 7

pi (*50 and 32 %, respectively) compared with unin-

fected mice, with levels returning to those in controls by

day 14 pi (Fig. 7a). Levels of SOD2 and SOD3 mRNA

were similar in infected and control mice on days 3 and 7

pi (Fig. 7b, c). However, on day 14 pi, the levels of SOD2

and SOD3 mRNA in CARV-infected mice showed an

increase (*100 % and 200 %, respectively) compared to

control mice (Fig. 7b, c).

Our observation that the expression of the SOD1, SOD2

and SOD3 genes showed alterations on different days

following CARV infection prompted us to test whether

viral infection could modify the activity of these AOEs.

The total SOD activity in CARV-infected mice was sig-

nificantly lower than in uninfected mice on day 3 pi

(*42 %), with levels returning to those in the control mice

by day 7 pi; however, the SOD activity was significantly

higher (*33 %) in CARV-infected mice on day 14 pi

(Fig. 8). Collectively, our data indicate that following

CARV infection, the livers of mice display lowered

abundance of the host antioxidant SOD, and this upregu-

lation appears to be due to a late event during disease

progression.

Table 1 Biomarkers of oxidative stress in control and CARV-infected BALB/c mice

Variable Experimental groups

Day 3 Day 7 Day 14

Control Infected Control Infected Control Infected

TBARS

(nmol/mg protein)

1.03 ± 0.41 1.02 ± 0.15 1.49 ± 0.27 1.49 ± 0.35 1.76 ± 0.49 1.66 ± 0.29

Carbonyl protein (nmol/mg protein) 3.98 ± 0.55 3.27 ± 0.94 3.57 ± 0.70 3.79 ± 1.68 3.54 ± 1.13 3.59 ± 0.93

TBARS, thiobarbituric acid reactive substances. Values are expressed as mean ± SD (n = 8). There were no significant differences between

control and infected groups

Fig. 6 Effect of CARV infection on the hepatic GSH content in

BALB/c mice. The GSH content was measured as described above in

the liver homogenates of CARV-infected and control mice on various

days after inoculation. The fold differences in GSH levels between

uninfected control mice and CARV-infected mice were determined.

The data are expressed as the mean ± standard deviation (n = 8 per

group). ** and *** indicate significant differences relative to the

control group at p \ 0.005 and p \ 0.0005, respectively

Changes in oxidative homeostasis in Caraparu virus infection

123

Discussion

The group C viruses were among the first arboviruses

discovered in the Brazilian Amazon Region during the

early 1950s. Geographically, these viruses occur in tropical

and subtropical areas of the Americas and have been

associated with human disease [13, 25, 34, 35]. CARV was

first isolated from a sentinel monkey in Utinga Forest in

Para State in northern Brazil [9]. Subsequently, it has been

isolated from patients reporting to clinics in the same

region, as well from a febrile human patient in Sao Paulo

State, in southeastern Brazil [19]. Yet, the epidemiological

characteristics, geographical range and pathogenesis of

CARV infection in humans remain unclear.

Previous studies have suggested that oxidative stress, as

part of the host cell response, might play an important role

in the pathogenesis of a variety of viral infections. In the

case of dengue virus infection, it was demonstrated that in

HepG2-grafted severe combined immunodeficiency dis-

ease (HepG2-grafted SCID) mice infected with dengue

virus type 2 (DENV2), supplementation with exogenous

GSH could inhibit oxidative stress effectively by reducing

oxidative damage to the organs, especially the liver [39].

Infection by Rift Valley fever virus, a member of the genus

Phlebovirus, family Bunyaviridae, causes an early decrease

in SOD1, and there is significant oxidative stress in the

infected cells [24]. Similarly, hepatitis B and C virus

infections cause extensive oxidative stress, which plays a

significant role in liver pathology [15].

In humans, CARV causes a systemic infection with the

clinical features of fever, chills and malaise [19, 34]. In this

study, we demonstrated that subcutaneous infection of

mice with CARV causes acute hepatitis and signs of sys-

temic disease such as ruffled fur, shivering, reduced food

intake and lethargy. We chose the subcutaneous route of

CARV inoculation because this is similar to the natural

route of viral infection. Up to now, there have been few

reports about oxidative stress in bunyavirus pathogenesis.

Although there is no information that CARV causes hep-

atitis in humans, we believe that the liver may be a

potential site for virus replication and therefore a very

Fig. 7 Alteration in the levels

of SOD1, SOD2 and SOD3

mRNA following infection by

CARV. The total RNAs from

livers of control and CARV-

infected BALB/c mice were

analyzed by q-RT-PCR with

primers for SOD1 (a), SOD2

(b) and SOD3 (c). The results

were normalized to GAPDH

RNA levels. The data are

expressed as the

mean ± standard deviation

(n = 8 per group). ** indicates

a significant difference relative

to the control group at

p \ 0.005. The letters a and b

indicate differences between

groups of CARV-infected

animals

Fig. 8 Alteration in the total SOD activity following infection by

CARV. A specific biochemical assay was used to determine the total

superoxide dismutase (SOD) activity in the livers of control and

CARV-infected mice. The data are expressed as the mean ± standard

deviation (n = 8 per group). * and **indicate significant differences

relative to the control group at p \ 0.05 and p \ 0.005, respectively.

The letters a and b indicate differences between the groups of CARV-

infected animals

F. C. Camini et al.

123

useful model for evaluating the involvement of oxidative

homeostasis on CARV pathology as well as the pathology

of other members of the family Bunyaviridae.

In the present study, CARV-infected mice developed

significant hepatic damage on the third and seventh days pi,

confirmed by histopathological studies and changes in the

serum levels of ALT/AST. CARV antigen was detected in

all infected animals on different days pi, in several areas of

the hepatic parenchyma and in the cytoplasm of the cells.

However, stronger staining was observed in the paren-

chyma of infected mice on day 3 pi when the CARV

progeny titer was 106 PFU per gram of liver, indicating that

CARV replication is highly efficient in the liver. Despite

the accentuated inflammation and liver damage observed

on the seventh day after CARV infection, the virus was not

recovered from the liver by titration. It is possible that the

extremely high level of inflammatory response observed on

day 7 pi is sufficient to inhibit viral replication. The

observed hepatotropic nature of CARV in our study sub-

stantiates earlier findings obtained upon intraperitoneal

inoculation of mice (B6C3F1), where the animals devel-

oped liver disease [3].

Next, we investigated whether CARV infection in mice

led to cellular oxidative stress in the liver. Elevation of the

levels of TBARS and carbonyl protein is an indicator of

oxidative damage, which is usually caused by an increase

in intracellular ROS. In this study, the levels of TBARS

and carbonyl protein in infected animals were similar to

those in control mice on different days, indicating that

oxidative-stress-induced damage of the liver does not

necessarily occur in CARV infection. Because GSH is the

major endogenous antioxidant produced by cells, we

investigated if CARV infection alters the GSH content in

the liver. There was an increase in hepatic GSH content in

the CARV-infected groups relative to the control groups on

all days pi. Thus, our study demonstrated that in CARV

infection, endogenous GSH might be involved in neutral-

izing ROS and constitute an important cellular system that

counteracts oxidation and plays a significant role in

maintaining a reductive intracellular environment.

Because it is essential to proper liver function, exoge-

nous GSH has been used for the inhibition of oxidative

stress. In a previous study using HepG2 cells, DENV2

infection resulted in decreased intracellular GSH levels,

and supplementation with GSH significantly decreased the

production of DENV2 [36]. Additionally, in mice infected

with DENV2, supplementation with GSH inhibited hepatic

oxidative stress [39]. In other studies, treatment with GSH

had an inhibitory effect on the production of influenza A

virus or herpes simplex virus type 1 (HSV-1), suppressed

rhinovirus-induced superoxide radical production, and

reduced the weights of the spleen and lymph node in HIV-

infected mice [5, 27–29]. These studies suggest that the

inhibition of oxidative pathways by antioxidants such as

GSH has therapeutic potential for viral diseases.

Because the other component of the cellular antioxidant

machinery is the SOD family of enzymes, we investigated

the roles of the intracellular isoforms SOD1/SOD2 and of

the extracellular isoform SOD3 in CARV infection. Levels

of SOD1 mRNA were decreased in CARV-infected mice

on days 3 and 7 pi, and levels of SOD2 and SOD3 mRNA

were higher in the infected mice on day 14 pi. Consistent

with the observed levels of SOD1, SOD2 and SOD3

mRNA, the total SOD activity in CARV-infected mice

relative to control mice was lower on day 3 pi and higher

on day 14 pi. Despite the reduction of cellular SOD activity

3 days after infection, there was an increase of approxi-

mately 20 % in GSH content on this day. SOD enzymes

convert O2- to H2O2, and the glutathione redox cycle is

complementary to catalase in converting H2O2 to water and

oxygen. In the presence of H2O2, reduced GSH can be

oxidized to its dimeric oxidized form, GSSH, protecting

cells from oxidative stress. The decrease in total SOD

activity 3 days after infection suggests that livers of

infected mice display lower levels of H2O2 at early time

points of infection. Then, when the amount of H2O2 is low,

the cells maintain high GSH content in part by preventing

the oxidation of GSH to GSSG.

Next, since recent studies have demonstrated that an

increase in TNF-a causes downregulation of SOD1

expression [1, 24], we investigated the relationship

between TNF-a and SOD1 after CARV infection. In

infected animals, there is a significant increase in TNF-amRNA levels 3 days after infection and a concomitant

decrease in SOD1 expression. Thus, our data suggest that

upregulated TNF-a could contribute to lowered SOD1

mRNA levels in CARV-infected mice during the early

stages of infection. TNF-a is a pleiotropic cytokine that

regulates a broad range of biological events, including cell

differentiation, proliferation, tissue development and death,

as well as inflammation and innate and adaptive immune

responses. The increase in the levels of TNF-a mRNA on

the third day after CARV infection was associated with

severity of clinical signs and inflammation in the liver of

BALB/c mice. Yet, this increase on the levels of TNF-apossibly activates different pathways of the innate immune

response, favoring the control of viral load, which may

result in resistance to CARV in BALB/c mice. However,

further studies are needed to better determine the role of

TNF-a in this model of CARV-induced liver disease.

Although our study was performed only on days 3, 7 and

14 after infection, it included a balanced representation of

the progression of the disease caused by CARV in the

livers of BALB/c mice from acute hepatitis until the

recuperation of the animals. However, assessing the effect

of the infection within 3 days could contribute to a better

Changes in oxidative homeostasis in Caraparu virus infection

123

understanding of the relationships between endogenous

GSH, ROS detoxification, other AOEs, and anti-inflam-

matory mediators on CARV-induced hepatic disease.

In relation to possible involvement of brain damage after

CARV infection, there are no data in the literature showing

that CARV is neurotropic after inoculation in adult mice.

According to Briton et al. [3], newborn mice develop

hepatitis and encephalitis after intracerebral injection of

CARV, but adult mice inoculated with virus by the intra-

peritoneal route develop only hepatic disease. Here, ani-

mals infected by the subcutaneous route with CARV

developed clinical signs and liver disease, without notice-

able neurological manifestations; however, possible dam-

age to the brain has not been investigated. It would be

interesting to examine, in future studies, if subcutaneous

infection by CARV in adult mice causes neurologic injury

and to investigate its relationship to oxidative homeostasis.

Based on our findings, we propose a model considering

the possible factors that might contribute to acute hepatitis

and disease resolution in CARV-infected BALB/c mice

(Fig. 9). Briefly, following subcutaneous infection with

105 PFU of CARV, the virus exhibits hepatic tropism.

Then, there is an increase in TNF-a levels at the early

stages of infection, which possibly activates the inflam-

matory response, leading to downregulation of SOD1

expression and favoring acute inflammation in the tissue

(with an increase in serum levels of AST/ALT), as well as

reduced SOD activity. However, despite the reduction in

SOD activity, no evident oxidative stress was observed. As

the infection progresses, an effective host response is

elicited in which TNF-a levels, inflammatory cells and

AST/ALT levels return to normal. There is also an increase

in SOD2 and SOD3 expression and total SOD activity,

which might help to control both virus load and disease

resolution. Furthermore, the increase in both the total SOD

activity and GSH content corroborate the hypothesis that

CARV infection may lead to increased levels of intracel-

lular O2-, which is metabolized to H2O2 by SOD, and GSH

detoxifies H2O2 by generating water and oxygen, main-

taining the oxidant/antioxidant balance and preventing

oxidative stress during CARV liver injury. However, fur-

ther work is needed to confirm this hypothesis, since levels

of ROS were not tested here.

The development of experimental models is a necessity

for virologists to understand pathogenesis. This report

describes a subcutaneous experimental infection of BALB/

c mice with CARV, resulting in hepatic disease with

alterations in antioxidant defenses, but no obvious oxida-

tive stress. Other important aspects to be investigated in

future studies comparing infections caused by CARV to

those caused by other hepatotropic pathogens include the

profiles of AOEs that are altered in severe versus mild

forms of hepatitis, the ability of each pathogen to trigger

the generation of ROS, and the extent and type of oxidative

damage. Collectively, our data shed light on some early

mechanisms that are operational in the murine host fol-

lowing exposure to CARV.

Acknowledgements This work received financial support from

Fundacao de Amparo a Pesquisa do Estado de Minas Gerais (FAP-

EMIG) – Process APQ-04125-10, Brazil. We thank Universidade

Federal de Ouro Preto (UFOP) and the Research Center in Biological

Sciences (NUPEB/UFOP), Brazil. The authors are grateful to col-

leagues from the Virus Laboratory (UFMG), Laboratory of Metabolic

Biochemistry (UFOP) and Experimental Nutrition (UFOP) for their

technical and scientific support. We are also grateful to Maria

Terezinha Bahia from Chagas’ Disease Laboratory (UFOP) for the

use of the real-time PCR ABI 7300 equipment (Applied Biosystems)

Subcutaneous infection (105 PFU)

CARV

ACUTE HEPATITIS

Effective host response

Virus replication

TNF- mRNA

AST/ALT

Inflammation

DISEASE RESOLUTION

No obvious oxidative damage

SOD1 mRNA

SOD activityLiver injuryGSH

Viral clearance

TNF- mRNA

AST/ALT

Inflammation

No obvious oxidative damage

SOD1/2/3 mRNA

SOD activityLiver recovery

LIVER

Hepatic tropism

H2O2(?)

H2O

GSH

Fig. 9 Schematic

representation of possible

factors associated with CARV

infection that contribute to the

acute hepatitis in BALB/c mice

and disease resolution

F. C. Camini et al.

123

and Jaquelline G. de Oliveira, Marcelo Eustaquio Silva, Melina Oli-

veira de Souza and Joamyr Victor Rossoni Junior for help with certain

experiments.

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