Immunology Letters 158 (2014) 126–133
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Callithrix penicillata: A feasible experimental model for dengue virus infection Milene Silveira Ferreira a,b , Paulo Henrique Gomes de Castro c , Gilmara Abreu Silva c , Samir Mansur Moraes Casseb a , Antônio Gregório Dias Júnior a , Sueli Guerreiros Rodrigues a , Raimunda do Socorro da Silva Azevedo a , Matheus Fernandes Costa e Silva d , Danielle Alves Gomes Zauli d , Márcio Sobreira Silva Araújo d , Samantha Ribeiro Béla d , Andréa Teixeira-Carvalho d , Olindo Assis Martins-Filho d,∗ , Pedro Fernando da Costa Vasconcelos a,e,∗∗ a
Instituto Evandro Chagas/SVS/MS, Ananindeua, Pará, Brazil Núcleo de Medicina Tropical Universidade Federal do Pará, Belém, Pará, Brazil c Centro Nacional de Primatas, Ananindeua, Pará, Brazil d Laboratório de Biomarcadores de Diagnóstico e Monitorac¸ão, Centro de Pesquisas René Rachou, Fundac¸ão Oswaldo Cruz-FIOCRUZ, Belo Horizonte, Brazil e Universidade do Estado do Pará, Belém, Pará, Brazil b
a r t i c l e
i n f o
Article history: Received 10 October 2013 Received in revised form 6 December 2013 Accepted 9 December 2013 Available online 17 December 2013 Keywords: Dengue virus infection Immune response Experimental model Non-human Primate
a b s t r a c t Although the murine models have the feasibility to reproduce some signs of dengue Virus (DENV) infection, the use of isogenic hosts with polarized immune response patterns does not reproduce the particularities of human disease. Our goal was to investigate the kinetics of peripheral blood biomarkers in immunocompetent Callithrix penicillata non-human primates subcutaneously infected with DENV3. The viral load of infected animals was determinated by quantitative real time PCR. Measurements of DENV-3/IgM were performed, and several parameters were assessed by hemogram: red blood cells count, hemoglobin, hematocrit, white blood cells count, neutrophils, monocytes, lymphocytes, and platelets count. The coagulogram was performed by prothrombin time (PT), and activated partial thromboplastin time (APTT) assays. The renal function was monitored by urea and creatinine, and the liver function by the aspartate (AST), and alanine (ALT) aminotransferases. Also, the level of the cytokines IL-6, TNF-␣, IL-2, IFN-␥, IL-4 and IL-5 was quantified during the experimental study. Data analysis was performed considering relevant differences when baseline fold changes were found outside from 0.75 to 1.5 range.
Abbreviations: DENV, dengue virus; dpi, days-post-infection; HIA, hemagglutination inhibition assay; APTT, activated partial thromboplastin time; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TNF-␣, tumor necrosis factor-alpha; IFN-␥, interferon-gamma; PT, prothrombin time; PLT, platelets; IgM, immunoglobulin M; IL-2, interleucin-2; IL-6, interleucin-6; IL-4, interleucin-4; IL-5, interleucin-5; DF, dengue fever; DHF, dengue hemorrhagic fever; DSS, dengue shock syndrome; IFA, immunofluorescence assay; CENP, Primate National Center; EDTA, ethylenediamine tetraacetic acid; RT-PCR, reverse transcriptase-PCR; qPCR, quantitative real time PCR; RBC, red blood cells; HGB, hemoglobin; HCT, hematocrit; NEU, neutrophils; MON, monocytes; LYM, lymphocytes; CREA, creatinine; MFI, mean fluorescence intensity; WBC, white blood cells. ∗ Corresponding author at: Laboratório de Biomarcadores de Diagnóstico e Monitorac¸ão, Centro de Pesquisas René Rachou, Fundac¸ão Oswaldo Cruz, Avenida Augusto de Lima, 1715, Barro Preto, Belo Horizonte, Minas Gerais 30 190-002, Brazil. Tel.: +55 31 3349 7764; fax: +55 31 3295 3115. ∗∗ Corresponding author at: Centro Colaborador da OMS/OPAS em Arbovírus, Departamento de Arbovirologia e Febres Hemorrágicas, Instituto Evandro Chagas, SVS/MS, Rodovia BR-316, km-7, 67030-000 Ananindeua, Pará, Brazil. Tel.: +55 91 3214 2271; fax: +55 91 3214 2299. E-mail addresses: [email protected] (M.S. Ferreira), [email protected] (P.H.G. de Castro), [email protected] (G.A. Silva), [email protected] (S.M.M. Casseb), [email protected] (A.G. Dias Júnior), [email protected] (S.G. Rodrigues), [email protected] (R.d.S.d.S. Azevedo), mfernandes@cpqrr.fiocruz.br (M.F.C.e. Silva), [email protected] (D.A.G. Zauli), sobreira@cpqrr.fiocruz.br (M.S.S. Araújo), samantha@cpqrr.fiocruz.br (S.R. Béla), andreat@cpqrr.fiocruz.br (A. Teixeira-Carvalho), daniellezauli@cpqrr.fiocruz.br (O.A. Martins-Filho), [email protected] (P.F.d.C. Vasconcelos). 0165-2478/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.imlet.2013.12.008
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Our data demonstrated that infected animals presented relevant signs of dengue disease, including peaks of viremia at 5 days-post-infection (dpi), peaks of anti-DENV-3 IgM at 15 dpi and hemaglutination inhibition assay (HIA) from 15 to at 60 dpi. Despite early monocytosis, slight neutrophilia and lymphocytosis, animals developed persistent leucopenia starting at 4 dpi. Anemia episodes were steady at 3–4 dpi. Patent thrombocytopenia was observed from 1 to 15 dpi with sporadic decrease of APTT. A substantial increase of ALT and AST was observed with higher peak at 4 dpi. Moreover, early increases of TNF-alpha and IFN-gamma besides late increase of IFN-gamma were observed. The analysis of biomarkers network pointed out two relevant strong axes during early stages of dengue fever, a protective axes TNF-alpha/Lymphocytes/Platelets, and a pathological IL-2/IL-6/Viremia/Monocyte/PT bond. Later on, the biomarker network highlighted the interaction IFN-gamma/PLT/DENV-3(IgM;HAI)/PT, and the involvement of type-2 cytokines (IL-4; IL-5). Our findings demonstrated that C. penicillata is a feasible experimental model for dengue virus infection, which could be useful to pathogenesis studies, discovery of novel antiviral drugs as well as to evaluate vaccine candidates against DENV. © 2014 Published by Elsevier B.V.
1. Introduction
2. Materials and methods
Dengue is an acute infectious disease caused by the dengue arbovirus (DENV), belonging to the Flaviviridae family, Flavivirus genus. The disease is transmitted to humans by Aedes mosquitoes, mainly Aedes aegypti, and is caused by one of the four antigenically distinct and genetically diverse viral serotypes (DENV-1, -2, -3, and -4). All four dengue serotypes circulate globally, and a relationship between disease severity and DENV serotype has been suggested [1]. Aside from asymptomatic infections, the disease presents three main clinical forms: dengue fever (DF), which is characterized by a non-specific febrile illness, dengue hemorrhagic fever (DHF) with or without shock syndrome known as dengue shock syndrome (DSS), which is characterized by capillary leakage, hemorrhagic events, and hypovolemic shock [2,3]. Recent studies stimate 392 million of dengue infections [4] each year, 96 millions of them presenting apparent infections with more than 2.5 billion people at risk of infection [5–7]. For both climatic and social reasons, the developing countries generally have high rate of A. aegypti infestation and remote possibilities for eradication of disease, which contributes to the rate of infection by arboviruses as occurs in Brazil [8]. Several immunocompetent, immunocompromised, and humanized mouse models have been developed to investigate the immunological, and clinical aspects of DENV infection [9–11]. Although these studies report that DENV-infected animals develop transient viremia, and produce antibodies, not all animals develop significant levels of viremia, and reproduce signs of disease [12–15]. Furthermore, while the murine models can reproduce some signs of human DF, the use of isogenic models presenting polarized immune response patterns does not reproduce the particularities of human disease. Others studies have established new animal models of DENV infection evaluating levels of viremia, hematological, and biochemical parameters of common marmosets (Callithrix jacchus) after DENV infection [16,17]. Recently, Yoshida et al. [18] observed that DENV infection in marmosets greatly induced responses of central memory CD4+ or CD8+ T cells subsets, and NKT cells. However, in these models, relevant immunological aspects involved in the immune response after DENV infection such as cytokine profile were not investigated. The absence of out-bred non-human primate model has postponed the understanding of the complexity of human DF pathogenesis. In fact, until the present date, there are no comprehensive information on DENV pathogenesis, and immunity in the existing non-murine experimental models that are able to mimic clinical and immunological aspects observed in DENV-infected humans. The goal of this study was to evaluate the feasibility of the C. penicillata non-human primate as an experimental model for DENV infection.
2.1. Ethical aspects This work was classified as a descriptive analytic controlled experimental study, and the samples were selected by convenience. The protocol was approved by the Ethics Committee on Animal Research at Evandro Chagas Institute – Primate National Center (IEC-CENP) – protocol #0061/2009, and by the System Authorization and Information on Biodiversity-SISBIO of Chico Mendes, Institute for Biodiversity Conservation-ICMBio protocol #220471. In this study, the viral sample of serotype DENV-3 (RNH 712 149) used belongs to the Hemorrhagic Fever and Arbovirus Section at Evandro Chagas Institute, and the authorization for use was obtained through the protocol #006031/2013-91. 2.2. DENV virus We used a viral sample of serotype DENV-3 (RNH 712 149) obtained from lymph node fragments from a fatal Dengue case classified by the Brazilian Ministry of Health as DHF/DSS. The virus was kept in C6/36 cell culture according to Igarashi, 1978, and a single additional passage was done before marmoset infections [19]. Immunofluorescence assay (IFA) was used to confirm the presence of DENV-3 in infected cells, according to Gubler et al. [20]. 2.3. Experimental infection in non-human primates The animals used in this study were selected from the C. penicillata colony at the CENP, located in Ananindeua-PA, Brazil. Twenty-two animals were selected and included in this study. The criteria used for selection were age between 1 and 10 years, and negative screening by hemagglutination inhibition assay (HIA) test for 23 different types of arboviruses including: Eastern Equine Encephalitis Virus (EEEV); Equine Encephalitis West Virus (WEEV); Mayaro Virus (MAYV); Mucambo Virus (MUCV); Guaroa Virus (GROV); Yellow Fever Virus (YFV); DENV-1, -2, -3, -4; Rocio Virus (ROCV); Ilheus Virus (ILHV); St. Louis Encephalitis Virus (SLEV); Tacaiuma Virus (TCMV); Maguari Virus (MAGV); Caraparu Virus (CARV); Oropouche Virus; Catu Virus (CATUV); Icoaraci Virus (ICOV); Utinga Virus (UTIV); Cacipacore Virus (CPCV); Bussuquara Virus (BSQV); Belém Virus (BLMV). All animals were infected subcutaneously with 500 L of cell suspension containing 1.45 × 104 DENV-3 viral particles/mL. The infective dose was calculated according to Malewiczt et al. [21]. The infected animals were kept in individual cages, observed daily, and have received water and food at libidum.
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Serological Profile
B
1.20
An-DENV-3
Virological Profile
↑
0.60 0.40
↑
D60
D45
D20
D7
D15
D6
D5
D4
D3
D2
-0.20
D1
0.00
20
Days aer Infecon
C ↑
DE NV - 1
500
Days aer infecon
Inverse of sera diluon
D60
D45
D20
D15
D7
D6
D5
D4
D3
D2
D1
0
Hemagglunaon Inhibion
5
200
-100 800
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DE NV -3 500
800 700 600 500 400 300 200 100 0 -100
D1 D2 D3 D4 D5 D6 D7 D15 D20 D45 D60
200
-100
800 700 600 500 400 300 200 100 0 -100
DE NV - 2
↑
D1 D2 D3 D4 D5 D6 D7 D15 D20 D45 D60
800
10
D EN V - 4
↑
D1 D2 D3 D4 D5 D6 D7 D15 D20 D45 D60
15
D1 D2 D3 D4 D5 D6 D7 D15 D20 D45 D60
# of Viral Parcles/mL
0.80
0.20
25
Viral Load
IgM Reacvity
A
OD (450nm)
1.00
Days aer infecon Fig. 1. Virological and serological profile of DENV-3 infected C. penicillata. (A) Viral load was assessed by qRT-PCR along infection; the results are expressed as number of viral DENV-3 particles/mL based on the standard curve as described in Section 2. (B) Anti-DENV-3 IgM reactivity was assayed in sera samples by MAC-ELISA along infection; the results are expressed as optical density (OD) as described in Section 2. (C) Hemagglutination inhibition assay (HIA) anti-DENV-1–4 antibodies were determined by visual observation of the goose red blood cell sedimentation. The results are expressed as reciprocal sera dilution as described in Section 2. Peaks of viremia, and serological reactivity are highlighted in the figure by arrows (↑).
2.4. Collection and storage of biological specimens Three whole blood samples collected in EDTA, sodium citrate or without anticoagulant were drawn from animals prior to DENV3 infection (day 0), and daily post-infection (dpi) starting at 1 dpi through 7 dpi, 15, 20, 45, and 60 dpi. Whole blood was collected, and processed for plasma and serum, and stored in duplicates at −20 ◦ C or −70 ◦ C until use. 2.5. Viral RNA isolation and RT-PCR Viral RNA used for RT-PCR assay was extracted from EDTA plasma samples by the PureLink® RNA Mini Kit (Ambion, Texas, USA) following the manufacturer’s protocol. Viral RNA was quantified using a Qubit® Fluorometric Quantitation (Invitrogen, California, USA), and commercial Qubit® RNA Assay Kit following the manufacturer’s protocol. Afterwards, viral RNA was used to synthesize cDNA using the EXPRESS One-Step Superscript® qRT-PCR Universal kit (Invitrogen, California, USA) according to Johnson et al. [22].
according to the manufacturer’s protocols. The competent bacterial cells (Escherichia coli strain TOP10F) were previously prepared by the method of calcium chloride (CaCl2 ) [23]. The plasmid DNA was extracted using a Miniprep DNA Purification System kit (Promega Corporation, Madison, Wisconsin, USA) following the manufacturer’s protocol. The concentration of recombinant plasmids containing DENV-3 inserts was determined using a Qubit 2.0 fluorometer (Invitrogen, California, USA) with a Qubit dsDNA BR Assay kit (Invitrogen, California, USA) following the manufacturer’s protocol. Serotype-specific DENV-3 primers (DENV-3F GGACTGGACACACGCACTCA, and DENV-3C CATGTCTCTACCTTCTCGACTTGTCT) were used according to Johnson et al. [22], and TaqMan qRT-PCR was also used during the assay. The RT-qPCR was performed using the ABI Prism 7500 Sequence Detection System (Applied Biosystem, California, USA) with a thermalcycling conditions set as follows: a cycle at 50 ◦ C for 2 min, followed by 45 cycles of 95 ◦ C for 10 min, 95 ◦ C for 15 s, and 56 ◦ C for 1 min. The viral load was expressed as number of DENV3 viral particles/mL based on the standard curve constructed using serial dilutions of plasmids containing the DENV-3 insert at concentrations ranging from 8 × 101 to 8 × 107 viral particles/mL.
2.6. Viral load determination by quantitative real time PCR (qPCR) 2.7. Serological analysis For the quantification of viral load, a standard curve was constructed using a plasmid provided by cloning of the amplicon using TOPO TA Cloning Kit (Invitrogen, California, USA),
Measurements of DENV-3 IgM were performed in sera samples by the MAC-ELISA method, according to Kuno et al. [24]. IgM
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Hematological Profile A
Leucócitos Totais
Leukogram
D60
D45
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D15
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D45
D20
D15
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D5
D4
0.00
↓
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↓
0.75
D3
APTT
1.50
D2
D45
D20
D15
D7
D6
D5
D4
D3
D2
↓ ↓ ↓
↓
D3
0.00
↓
3.00 2.25
D2
↓ ↓
↓
0.75
0.00
1.50
0.75
1.50 0.75
D1
↓
1.50
Hematocrit
D60
D45
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D7
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D5
D4
D3
D2
2.25
0.00
0.00
↓
D2
↓ ↓
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D1
↓
D1
Platelets D60
PT
Hemoglobin
D45
D20
D7
D15
D6
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D2
D1
Red Blood Cells
D45
D20
D7
D6
D5
D4
D3
D2
D1
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D45
D20
2.25
1.50
2.25 D1
Neutrophils
D15 D15
D7
D6
D5
D4
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D1
↓↓
↓ ↓↓ ↓ ↓ ↓↓ ↓
3.00
↓
Monocytes
↓
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0.75
1.50 0.75
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Hemácia Erythrogram 2.25
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↓
0.75
Coagulogram
C
↓
White Blood Cells
B
↓
1.50
0.00
Baseline fold changes / DENV-3 pre-infecon
↓
2.25
Days aer infecon
Fig. 2. Hematological profile of DENV-3 infected C. penicillata. (A) Leukogram was measured by neutrophil, monocyte, and lymphocyte counts. (B) Erythrogram was measured by red blood cell count, hemoglobin, and hematrocrit determinations. (C) Coagulogram was measured by platelet counts, prothrombin time (PT), and activated partial thromboplastin time (APTT) assays. All data are expressed as baseline fold changes relative to DENV-3 pre-infection. Relevant differences are highlighted by arrows in the figure (↓ = decrease at 1.5 baseline folds).
reactivity was expressed as optical density (OD) at 450 nm. The HIA test was performed as described by Clarke et al. [25], and adapted to microplate by Shope [26], and covered 23 different arboviruses as mentioned in Section 2.3. These data were used to analyze the occurrence of cross-reactivity between the evaluated arboviruses. The results were determined by visual observation of the goose red blood cell sedimentation, and were expressed as reciprocal sera dilution.
2.9. Biochemical studies The renal function was monitored by urea and creatinine (CREA) levels, and the liver function by the aspartate (AST) and alanine (ALT) aminotransferases. The tests were performed using commercial kits (ROCHE, São Paulo, Brazil) in a biochemical analyzer (COBAS MIRA PLUS 400, São Paulo, Brazil), according to the manufacture’s instructions. The results were expressed as IU/mL.
2.8. Hemogram and coagulogram The hemogram was performed in EDTA blood samples using MS4 Hematology Analyzer (Melet Schloesing Laboratoires, France), according to the manufacture’s protocol, and included the following parameters: red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), white blood cells (WBC), neutrophils (NEU), monocytes (MON), lymphocytes (LYM), and platelets (PLT). The coagulogram was performed in sodium citrate plasma samples to measure prothrombin time (PT) and activated partial thromboplastin time (APTT) assays using a CLOTimer coagulometer (Clot, São Paulo, Brazil), as described by the manufacturer.
2.10. Quantification of serum cytokines The level of cytokines (IL-6, TNF-␣, IL-2, IFN-␥, IL-4 and IL-5) was quantified in sera collected from DENV-3 infected marmosets during the experimental study. We used the system Cytometric Bead Array (CBA) (BD Pharmingen, California, USA) according to manufacturer’s protocol. The assay was analyzed using a FACScaliburTM flow cytometer equipped with four color detection system, and the CELLQuestTM software (Becton Dickinson, California, USA) to perform data acquisition. The data analysis was performed using BD CBA software (Becton Dickinson). The results were expressed as mean fluorescence intensity (MFI).
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2.11. Data analysis
Biochemical Profile
3.75 3.00
9.75
9.00 8.25 7.50 6.75
6.00
6.75
6.00
↓
D20
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D20
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↓
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↓ D15 D20
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2.25
1.50 0.75 0.00
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3.00
D6 D7
5.25 4.50 3.75
7.50
AST
D60
D7
D6 D6
D15
D5 D5
D15
D4 D4
D7
D2
D3 D3
D1 D1
0.00
↓
D1
Baseline foldchanges / DENV-3pre-infecon
0.75
↓
ALT
1.50
↓
To assess the kinetics of hepatic and renal functions of DENV-3 infected C. penicillata, the serum levels of AST and ALT as well as urea/CREA, respectively, were measured in serum samples along the infection (Fig. 3). Our results showed an early increase in
2.25
↓
Creanine
3.00
↓
3.3. Increased levels of ALT and AST with higher peak at 4 dpi with a persistent decrease of urea along the infection were the major serological markers of DENV-3 infection course in C. penicillata
↓↓
↓ ↓ ↓ ↓↓
↓
Hematological profile of DENV-3 infected C. penicillata was carried out in EDTA whole blood samples. Leukogram, erythrogram and coagulogram data were expressed as baseline fold changes relative to the DENV-3 pre-infection profile. Data analysis demonstrated that despite an early monocytosis at 1 dpi along with a slight neutrophilia at 2 dpi, lymphocytosis at 3 and 60 dpi, and marked decrease in the WBC was observed along the infection at 4, 6, 7, 15, and 45 dpi (Fig. 2A). Our data also demonstrated a decrease in the RBC counts at 3–4 dpi, with clear anemia episodes at 3 and 4 dpi, as demonstrated by lower hemoglobin, and hematocrit values (Fig. 2B). A patent thrombocytopenia was observed along the infection at days 1–15, with sporadic decreases in the APTT values at 2, 6, and 60 dpi (Fig. 2C).
↓
↓
D2
Urea
0.00
3.1. Peak of viremia at 5 dpi along with high anti-DENV-3 IgM reactivity at 15 dpi, and maximum of antibodies levels from 15 to 60 dpi were virological/serological biomarkers in DENV-3 infected C. penicillata
3.2. Despite an early monocytosis at 1 dpi, slight neutrophilia at 2 dpi, and lymphocytosis at 3 dpi, DENV-3 infected C. penicillata developed a persistent leucopenia from 4 to 45 dpi
1.50 0.75
3. Results
Viral load was determinated by RT-qPCR in serum samples of DENV-3 infected C. penicillata throughout the assessment period (1–60 dpi). Our findings demonstrated that virus could be detected as early as 2 dpi with an outstanding peak of viremia at 5 dpi (Fig. 1A). Analysis of the anti-DENV-3 serological reactivity was performed throughout the assessment period (1–60 dpi) by IgM MAC-ELISA as well as by HAI. Our data showed that anti-DENV-3 IgM reached the higher reactivity peak at 15 dpi (Fig. 1B), whereas the maximum of HAI reactivity was achieved from 15 to 60 dpi with a clear cross-reactivity profile amongst DENV serotypes 1–4 (Fig. 1C).
2.25
↓
All data sets, except virological and serological results, were transformed into baseline fold ratio prior to analysis. Baseline fold value was obtained for each parameter as the ratio between the values observed after DENV-3 infection by the baseline value observed at DENV-3 pre-infection. Data analysis was performed considering relevant differences when baseline fold changes were found outside from 0.75 to 1.5 range (gray zones in figures), i.e. relevant decrease when the baseline fold change is lower than 0.75, and relevant increase when the baseline fold change is higher than 1.5. A matrix with all measured biomarkers, expressed as baseline fold changes relative to DENV-3 pre-infection was assembled, and used to calculate the Spearman’s rank correlation indexes. Significant correlations at p < 0.05 indicating interactivity amongst biomarkers were selected to design biomarker networks using open source software (Cytoscape) [27].
↓
130
Days aerinfecon Fig. 3. Biochemical profile of hepatic and renal function of DENV-3 infected C. penicillata. Serum levels of urea, creatinine (CREA), alanine (ALT), and aspartate (AST) aminotransferases are expressed as baseline fold changes relative to DENV-3 preinfection. Relevant differences are highlighted by arrows in the figure (↓ = decrease at 1.5 baseline folds).
the AST levels at 1–4, and 6 dpi with a clear increase in the ALT levels at 2–6 and 7 dpi. Moreover, a persistent decrease in the urea levels was observed with minor changes in the CREA levels (Fig. 3).
3.4. Early increase in TNF-˛ and IFN- levels at 3 dpi with later increases of IFN- at 6, and 15 dpi were found along the DENV-3 infection course in C. penicillata Quantification of serum pro-inflammatory (IL-6, TNF-␣, IL-2, IFN-␥), and type-2 cytokines (IL-4 and IL-5) was carried out (Fig. 4). The results pointed out to increased levels of TNF-␣ and IFN-␥ at 3 dpi, along with later increase of IFN-␥ levels at 6, and 15 dpi (Fig. 4). Isolated decreases in TNF-␣ at 45 dpi, and IL-5 at 7 dpi were also reported (Fig. 4).
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Cytokine Profile
D45
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0.75
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0.75
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1.50
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2.25
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↑
IL-5
2.25
D2
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0.75
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IL-2
IFN-γ
↑
D15
1.50
D15
1.50
D7
2.25
D7
2.25
D6
D60
D45
D20
D7
D15
D6
D5
D4
D3
D2
0.00
D1
0.00
↓
D4
IL-6
0.75
IL-4
Baseline foldchanges / DENV-3pre-infecon
0.75
D3
1.50
↑
D2
1.50
D1
2.25
TNF-α
2.25
Days aer infecon Fig. 4. Kinetic profile of serum cytokines in DENV-3 infected C. penicillata. Serum cytokines (IL-6, TNF-␣, IL-2, IFN-␥, IL-4, and IL-5) were detected by cytometric bead array (CBA) as described in Section 2. The results are expressed as baseline fold changes relative to DENV-3 pre-infection. Relevant differences are highlighted by arrows in the figure (↓ = decrease at 1.5 baseline folds).
3.5. Two outstanding cytokine network axes (IL-6/IL-2/Viremia/MON/PT, and TNF-˛/LYM/PTL) were reported during early DENV-3 infection in C. penicillata Aiming to assess the interactivity and conections amongst the several biomarkers inclued in our investigation, we have assembled biological networks observed following early and late DENV-3 infection course in C. penicillata (Fig. 5). Our results demonstrated that during early DENV-3 infection, strong correlation axes could be observed in C. penicillata peripheral blood biomarkers, including two major axes. One axes was composed by strong link between IL-6/IL-2/Viremia/MON/PT. Moreover, another axes with strong correlation could be observed between TNF-a/LYM/PTL (Fig. 5 – left network). 3.6. Interactions between IFN-/PLT/DENV-3(IgM;HIA)/PT, and the involvement of type-2 cytokines (IL-4; IL-5) were the major findings of later stages of DENV-3 infection course in C. penicillata The interactivity and connections amongst the biomarkers during the later stages of DENV-3 infection in C. penicillata are shown in Fig. 5. Our data demonstrated that weaker but multiples correlation axes could be identified between C. penicillata peripheral blood biomarkers, including several axes of negative correlation. Interactions between IFN-␥/PLT/DENV-3(IgM;HIA) with negative
correlation with PT could be observed. Furthermore, another axes involving type-2 cytokines (IL-4; IL-5) could also be identified (Fig. 5 – right network).
4. Discussion Dengue is a mosquito-borne viral disease of expanding geographical range and incidence, and is considered the most important and widespread disease caused by arbovirus worldwide, affecting more than 100 countries. Infection by one of the four serotypes of DENV induces a spectrum of disease manifestations, ranging from asymptomatic to life-threatening DHF/DSS. Many efforts have been made to elucidate several aspects of DENVinduced disease, but its pathogenesis is complex and remains unclear. Understanding the mechanisms involved in the early stages of infection is crucial to determine and develop safe therapeutics to prevent the severe outcomes of disease without interfering with control of infection [26]. A common major technical barrier in understanding the pathogenesis of DENV infection is the absence of a suitable animal model that mimics completely dengue disease [28,29]. This fact is justified, for example, by the limited infectivity and modest DENV replication observed in murine models. The purpose of our study was
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Fig. 5. Biomarker networks in DENV-3 infected C. penicillata. Biomarker network were assembled early 1–7 (left panel), and late 15–60 (right panel) following DENV-3 infection according to baseline changes. Node patterns are used to indicate decreased (white dashed), increased (black), oscillating (black dashed) or stable (white) values. Correlations between biomarker pairs are represented by continuous (positive) or dashed (negative) lines. The strength of the correlations is represented by thin line (moderate, r = 0.36–0.67) or thick line (strong, r = 0.68–1.00), according to Taylor [31]. WBC – white blood cells; NEU – neutrophils; MON – monocytes; LYM – lymphocytes; RBC – red blood cells; HCT – hematocrit; HB – hemoglobin; PLT – platelets; PT – prothrombin time; APPT – activated partial thromboplastin time; CREA – creatinine; ALT – alanine aminotransferase; AST – aspartate aminotransferase; VL – viral load; IgM – DENV-3 IgM; HIA – hemmaglutination inhibition assay IgM/IgG.
to develop a feasible experimental model to study dengue virus infection using the C. penicillata non-human primate. Recently, Omatsu et al. [16] performed studies with marmosets of Callithrix jacchus species infected with one of four DENV serotypes isolated from clinical cases of dengue fever in humans, and the establishment of infection was observed after the primary DENV inoculation. The viral RNA was detected in all infected marmosets. Anti-DENV-1, anti-DENV-2, anti-DENV-3 IgM antibodies were detected at 5 dpi, and anti-DENV-4 IgM antibodies were detected at 7 dpi. In this study, a peak of viremia was found at day 5 after DENV-3 infection. Several research studies in humans describe seroepidemiological cross-reactivity between the four serotypes of DENV, with other viruses belonging to Flavivirus genus in DENV infected individuals [30]. In our current study, there was cross-reactivity between the DENV-1, -2 and -4 serotypes. Onlamoon et al. [14] carried out a complete blood cell count in DENV-infected rhesus monkeys. During acute infection, analysis of the erythrogram indicated a slight decrease in the hemoglobin levels, a significant reduction in the hematocrit values, and no changes in the number of RBC. The WBC count showed a significant leukopenia at days 1, 3, 5, 7, 10 after DENV-3 challenge, and leukocytosis at day 14 dpi. Similar to the findings of Onlamoon et al. [14], our study found a slight decrease in hemoglobin and hematocrit values during acute infection caused by DENV-3. Similarly, it was also demonstrated leukopenia caused by a decreased frequency of neutrophils, monocytes, and lymphocytes after DENV-3 infection. Leukopenia may have been caused by a decrease in neutrophil counts, and this alteration is commonly reported in DENV infected individuals, regardless of serotype [32]. Moreover, in our study, we have also observed a slight lymphocytosis at the end of the acute phase, and an early monocytosis at 1 dpi. Our results still showed a modest increase in the levels of AST and ALT, and a significant increase in the APTT during the acute phase. Thrombocytopenia was also observed at 1–15 dpi. Approximately, one-third of DF patients may
have mild hemorrhage manifestations, which may be related to the low platelet count [2]. In this context, thrombocytopenia was an outstanding finding observed in the first days after infection by DENV-3 in marmosets. This finding was similar with those reports found in both humans and rhesus monkeys [14,31]. Indeed, thrombocytopenia was indicative of infection in the experimental model proposed by our study. The immune response after DENV infection is a significant factor associated with disease severity, and induction of symptoms [1]. One of the main components of the immune system against DENV infection includes interferons (IFNs). Gonc¸alves et al. [2] have found that C57BL/6 mice infected with 7.2 × 107 PFU of DENV-1 – strain Mochizuki showed a significant increase of IFN-␥, with peak at 10 dpi. In the current study, we observed a significant increase of IFN-␥ at 3, 6 and 20 dpi. The proinflammatory cytokines seem to play a pathological role during host response to DENV infection. For example, increased levels of TNF-␣ have been associated with severity of dengue manifestation in humans [28]. Similar finding was detected in DENV-3 infected marmosets, which showed a significant increase of TNF-␣ at 3 dpi. The analysis of biomarkers network pointed out two relevant strong axes during early stages of dengue fever, a protective axes TNF-␣/Lymphocytes/Platelets, and a pathological IL-2/IL6/Viremia/Monocyte/PT bond. Later on, the biomarker network highlighted the interaction IFN-␥/PLT/DENV-3(IgM;HIA)/PT, and the involvement of type-2 cytokines (IL-4; IL-5). 5. Conclusions Our data demonstrated that C. penicillata is a feasible experimental model for DENV infection, because it was able to reproduce many aspects of DENV disease in terms of viremia, serological profile, liver damage, relevant alterations in hematological, biochemical, and immunological parameters. Therefore, this model
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could be useful to pathogenesis studies, discovery of novel antiviral drugs as well as to evaluate vaccine candidates against DENV. Competing interests The author(s) declare that they have no competing interests. Acknowledgments This study was supported by grants from Fundac¸ão Oswaldo Cruz (FIOCRUZ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundac¸ão de Amparo à Pesquisa de Minas Gerais (FAPEMIG), and Instituto Nacional de Ciência e Tecnologia para Febres Hemorrágicas Virais (INCT-FHV grant CNPq/CAPES/FAPESPA 573739/2008-0). The authors thank the program for technological development in tools for health-PDTISFIOCRUZ for the use of its facilities. ATC, OAMF, and PFCV thank CNPq for fellowships (PQ); MSF was supported by a fellowship of CAPES. MSF, PHGC, GAS, SMMC, AGDJ, SGR, RSSA, and PFCV designed the study, and performed the experiments; MFCS, DAGZ, SRB, ATC, and OAMF wrote the paper. All authors read and approved the final manuscript. References [1] Herrero LJ, Zakhary A, Gahan ME, Nelson MA, Herring BL, Hapel AJ, et al. Dengue virus therapeutic intervention strategies based on viral, vector and host factors involved in disease pathogenesis. Pharmacol Ther 2013;137: 266–82. [2] Gonc¸alves D, de Queiroz Prado R, Almeida Xavier E, Cristina de Oliveira N, da Matta Guedes PM, da Silva JS, et al. Imunocompetent mice model for dengue virus infection. SciWorld J 2012;2012:1–12. [3] Yacoub S, Mongkolsapaya J, Screaton G. The pathogenesis of dengue. Curr Opin Infect Dis 2013;29:7221–8. [4] Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, et al. The global distribution and burden of dengue. Nature 2013;25:504–7. [5] Gubler DJ. Dengue and dengue hemorrhagic fever: its history and resurgence as a global public health problem. In: Gubler DJ, Kuno G, editors. Dengue and dengue hemorrhagic fever. London: Cab International; 1997. [6] Murray PR, Rosenthal KS, Kobayashi GS, Pfaller MA. Microbiologia médica. 4th ed. Rio de Janeiro: Guanabara Koogan; 2000. [7] Guzman MG, Alvarez M, Halstead SB. Secondary infection as a risk factor for dengue hemorrhagic fever/dengue shock syndrome: an historical perspective and role of antibody-dependent enhancement of infection. Arch Virol 2013:1–15. [8] BRASIL. Ministério da Saúde Guia de vigilância epidemiológica. Brasília: Fundac¸ão Nacional de Saúde; 2002. [9] Chen WF, Yeh WT, Yang MY. A model of the real-time correlation of viral titers with immune reactions in antibody-dependent enhancement of dengue2 infection. FEMS Immunol Med Microbiol 2001;30:1–7. [10] Kuruvilla JG, Troyer RM, Devi S, Akkina R. Dengue virus infection and immune response in humanized RAG2(−/−)gamma(c)(−/−) (RAG-hu) mice. Virology 2007;369:143–52.
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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Callithrix penicillata: A feasible experimental model for dengue virus infection Milene Silveira Ferreira a,b , Paulo Henrique Gomes de Castro c , Gilmara Abreu Silva c , Samir Mansur Moraes Casseb a , Antônio Gregório Dias Júnior a , Sueli Guerreiros Rodrigues a , Raimunda do Socorro da Silva Azevedo a , Matheus Fernandes Costa e Silva d , Danielle Alves Gomes Zauli d , Márcio Sobreira Silva Araújo d , Samantha Ribeiro Béla d , Andréa Teixeira-Carvalho d , Olindo Assis Martins-Filho d,∗ , Pedro Fernando da Costa Vasconcelos a,e,∗∗ a
Instituto Evandro Chagas/SVS/MS, Ananindeua, Pará, Brazil Núcleo de Medicina Tropical Universidade Federal do Pará, Belém, Pará, Brazil c Centro Nacional de Primatas, Ananindeua, Pará, Brazil d Laboratório de Biomarcadores de Diagnóstico e Monitorac¸ão, Centro de Pesquisas René Rachou, Fundac¸ão Oswaldo Cruz-FIOCRUZ, Belo Horizonte, Brazil e Universidade do Estado do Pará, Belém, Pará, Brazil b
a r t i c l e
i n f o
Article history: Received 10 October 2013 Received in revised form 6 December 2013 Accepted 9 December 2013 Available online 17 December 2013 Keywords: Dengue virus infection Immune response Experimental model Non-human Primate
a b s t r a c t Although the murine models have the feasibility to reproduce some signs of dengue Virus (DENV) infection, the use of isogenic hosts with polarized immune response patterns does not reproduce the particularities of human disease. Our goal was to investigate the kinetics of peripheral blood biomarkers in immunocompetent Callithrix penicillata non-human primates subcutaneously infected with DENV3. The viral load of infected animals was determinated by quantitative real time PCR. Measurements of DENV-3/IgM were performed, and several parameters were assessed by hemogram: red blood cells count, hemoglobin, hematocrit, white blood cells count, neutrophils, monocytes, lymphocytes, and platelets count. The coagulogram was performed by prothrombin time (PT), and activated partial thromboplastin time (APTT) assays. The renal function was monitored by urea and creatinine, and the liver function by the aspartate (AST), and alanine (ALT) aminotransferases. Also, the level of the cytokines IL-6, TNF-␣, IL-2, IFN-␥, IL-4 and IL-5 was quantified during the experimental study. Data analysis was performed considering relevant differences when baseline fold changes were found outside from 0.75 to 1.5 range.
Abbreviations: DENV, dengue virus; dpi, days-post-infection; HIA, hemagglutination inhibition assay; APTT, activated partial thromboplastin time; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TNF-␣, tumor necrosis factor-alpha; IFN-␥, interferon-gamma; PT, prothrombin time; PLT, platelets; IgM, immunoglobulin M; IL-2, interleucin-2; IL-6, interleucin-6; IL-4, interleucin-4; IL-5, interleucin-5; DF, dengue fever; DHF, dengue hemorrhagic fever; DSS, dengue shock syndrome; IFA, immunofluorescence assay; CENP, Primate National Center; EDTA, ethylenediamine tetraacetic acid; RT-PCR, reverse transcriptase-PCR; qPCR, quantitative real time PCR; RBC, red blood cells; HGB, hemoglobin; HCT, hematocrit; NEU, neutrophils; MON, monocytes; LYM, lymphocytes; CREA, creatinine; MFI, mean fluorescence intensity; WBC, white blood cells. ∗ Corresponding author at: Laboratório de Biomarcadores de Diagnóstico e Monitorac¸ão, Centro de Pesquisas René Rachou, Fundac¸ão Oswaldo Cruz, Avenida Augusto de Lima, 1715, Barro Preto, Belo Horizonte, Minas Gerais 30 190-002, Brazil. Tel.: +55 31 3349 7764; fax: +55 31 3295 3115. ∗∗ Corresponding author at: Centro Colaborador da OMS/OPAS em Arbovírus, Departamento de Arbovirologia e Febres Hemorrágicas, Instituto Evandro Chagas, SVS/MS, Rodovia BR-316, km-7, 67030-000 Ananindeua, Pará, Brazil. Tel.: +55 91 3214 2271; fax: +55 91 3214 2299. E-mail addresses: [email protected] (M.S. Ferreira), [email protected] (P.H.G. de Castro), [email protected] (G.A. Silva), [email protected] (S.M.M. Casseb), [email protected] (A.G. Dias Júnior), [email protected] (S.G. Rodrigues), [email protected] (R.d.S.d.S. Azevedo), mfernandes@cpqrr.fiocruz.br (M.F.C.e. Silva), [email protected] (D.A.G. Zauli), sobreira@cpqrr.fiocruz.br (M.S.S. Araújo), samantha@cpqrr.fiocruz.br (S.R. Béla), andreat@cpqrr.fiocruz.br (A. Teixeira-Carvalho), daniellezauli@cpqrr.fiocruz.br (O.A. Martins-Filho), [email protected] (P.F.d.C. Vasconcelos). 0165-2478/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.imlet.2013.12.008
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Our data demonstrated that infected animals presented relevant signs of dengue disease, including peaks of viremia at 5 days-post-infection (dpi), peaks of anti-DENV-3 IgM at 15 dpi and hemaglutination inhibition assay (HIA) from 15 to at 60 dpi. Despite early monocytosis, slight neutrophilia and lymphocytosis, animals developed persistent leucopenia starting at 4 dpi. Anemia episodes were steady at 3–4 dpi. Patent thrombocytopenia was observed from 1 to 15 dpi with sporadic decrease of APTT. A substantial increase of ALT and AST was observed with higher peak at 4 dpi. Moreover, early increases of TNF-alpha and IFN-gamma besides late increase of IFN-gamma were observed. The analysis of biomarkers network pointed out two relevant strong axes during early stages of dengue fever, a protective axes TNF-alpha/Lymphocytes/Platelets, and a pathological IL-2/IL-6/Viremia/Monocyte/PT bond. Later on, the biomarker network highlighted the interaction IFN-gamma/PLT/DENV-3(IgM;HAI)/PT, and the involvement of type-2 cytokines (IL-4; IL-5). Our findings demonstrated that C. penicillata is a feasible experimental model for dengue virus infection, which could be useful to pathogenesis studies, discovery of novel antiviral drugs as well as to evaluate vaccine candidates against DENV. © 2014 Published by Elsevier B.V.
1. Introduction
2. Materials and methods
Dengue is an acute infectious disease caused by the dengue arbovirus (DENV), belonging to the Flaviviridae family, Flavivirus genus. The disease is transmitted to humans by Aedes mosquitoes, mainly Aedes aegypti, and is caused by one of the four antigenically distinct and genetically diverse viral serotypes (DENV-1, -2, -3, and -4). All four dengue serotypes circulate globally, and a relationship between disease severity and DENV serotype has been suggested [1]. Aside from asymptomatic infections, the disease presents three main clinical forms: dengue fever (DF), which is characterized by a non-specific febrile illness, dengue hemorrhagic fever (DHF) with or without shock syndrome known as dengue shock syndrome (DSS), which is characterized by capillary leakage, hemorrhagic events, and hypovolemic shock [2,3]. Recent studies stimate 392 million of dengue infections [4] each year, 96 millions of them presenting apparent infections with more than 2.5 billion people at risk of infection [5–7]. For both climatic and social reasons, the developing countries generally have high rate of A. aegypti infestation and remote possibilities for eradication of disease, which contributes to the rate of infection by arboviruses as occurs in Brazil [8]. Several immunocompetent, immunocompromised, and humanized mouse models have been developed to investigate the immunological, and clinical aspects of DENV infection [9–11]. Although these studies report that DENV-infected animals develop transient viremia, and produce antibodies, not all animals develop significant levels of viremia, and reproduce signs of disease [12–15]. Furthermore, while the murine models can reproduce some signs of human DF, the use of isogenic models presenting polarized immune response patterns does not reproduce the particularities of human disease. Others studies have established new animal models of DENV infection evaluating levels of viremia, hematological, and biochemical parameters of common marmosets (Callithrix jacchus) after DENV infection [16,17]. Recently, Yoshida et al. [18] observed that DENV infection in marmosets greatly induced responses of central memory CD4+ or CD8+ T cells subsets, and NKT cells. However, in these models, relevant immunological aspects involved in the immune response after DENV infection such as cytokine profile were not investigated. The absence of out-bred non-human primate model has postponed the understanding of the complexity of human DF pathogenesis. In fact, until the present date, there are no comprehensive information on DENV pathogenesis, and immunity in the existing non-murine experimental models that are able to mimic clinical and immunological aspects observed in DENV-infected humans. The goal of this study was to evaluate the feasibility of the C. penicillata non-human primate as an experimental model for DENV infection.
2.1. Ethical aspects This work was classified as a descriptive analytic controlled experimental study, and the samples were selected by convenience. The protocol was approved by the Ethics Committee on Animal Research at Evandro Chagas Institute – Primate National Center (IEC-CENP) – protocol #0061/2009, and by the System Authorization and Information on Biodiversity-SISBIO of Chico Mendes, Institute for Biodiversity Conservation-ICMBio protocol #220471. In this study, the viral sample of serotype DENV-3 (RNH 712 149) used belongs to the Hemorrhagic Fever and Arbovirus Section at Evandro Chagas Institute, and the authorization for use was obtained through the protocol #006031/2013-91. 2.2. DENV virus We used a viral sample of serotype DENV-3 (RNH 712 149) obtained from lymph node fragments from a fatal Dengue case classified by the Brazilian Ministry of Health as DHF/DSS. The virus was kept in C6/36 cell culture according to Igarashi, 1978, and a single additional passage was done before marmoset infections [19]. Immunofluorescence assay (IFA) was used to confirm the presence of DENV-3 in infected cells, according to Gubler et al. [20]. 2.3. Experimental infection in non-human primates The animals used in this study were selected from the C. penicillata colony at the CENP, located in Ananindeua-PA, Brazil. Twenty-two animals were selected and included in this study. The criteria used for selection were age between 1 and 10 years, and negative screening by hemagglutination inhibition assay (HIA) test for 23 different types of arboviruses including: Eastern Equine Encephalitis Virus (EEEV); Equine Encephalitis West Virus (WEEV); Mayaro Virus (MAYV); Mucambo Virus (MUCV); Guaroa Virus (GROV); Yellow Fever Virus (YFV); DENV-1, -2, -3, -4; Rocio Virus (ROCV); Ilheus Virus (ILHV); St. Louis Encephalitis Virus (SLEV); Tacaiuma Virus (TCMV); Maguari Virus (MAGV); Caraparu Virus (CARV); Oropouche Virus; Catu Virus (CATUV); Icoaraci Virus (ICOV); Utinga Virus (UTIV); Cacipacore Virus (CPCV); Bussuquara Virus (BSQV); Belém Virus (BLMV). All animals were infected subcutaneously with 500 L of cell suspension containing 1.45 × 104 DENV-3 viral particles/mL. The infective dose was calculated according to Malewiczt et al. [21]. The infected animals were kept in individual cages, observed daily, and have received water and food at libidum.
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Days aer infecon Fig. 1. Virological and serological profile of DENV-3 infected C. penicillata. (A) Viral load was assessed by qRT-PCR along infection; the results are expressed as number of viral DENV-3 particles/mL based on the standard curve as described in Section 2. (B) Anti-DENV-3 IgM reactivity was assayed in sera samples by MAC-ELISA along infection; the results are expressed as optical density (OD) as described in Section 2. (C) Hemagglutination inhibition assay (HIA) anti-DENV-1–4 antibodies were determined by visual observation of the goose red blood cell sedimentation. The results are expressed as reciprocal sera dilution as described in Section 2. Peaks of viremia, and serological reactivity are highlighted in the figure by arrows (↑).
2.4. Collection and storage of biological specimens Three whole blood samples collected in EDTA, sodium citrate or without anticoagulant were drawn from animals prior to DENV3 infection (day 0), and daily post-infection (dpi) starting at 1 dpi through 7 dpi, 15, 20, 45, and 60 dpi. Whole blood was collected, and processed for plasma and serum, and stored in duplicates at −20 ◦ C or −70 ◦ C until use. 2.5. Viral RNA isolation and RT-PCR Viral RNA used for RT-PCR assay was extracted from EDTA plasma samples by the PureLink® RNA Mini Kit (Ambion, Texas, USA) following the manufacturer’s protocol. Viral RNA was quantified using a Qubit® Fluorometric Quantitation (Invitrogen, California, USA), and commercial Qubit® RNA Assay Kit following the manufacturer’s protocol. Afterwards, viral RNA was used to synthesize cDNA using the EXPRESS One-Step Superscript® qRT-PCR Universal kit (Invitrogen, California, USA) according to Johnson et al. [22].
according to the manufacturer’s protocols. The competent bacterial cells (Escherichia coli strain TOP10F) were previously prepared by the method of calcium chloride (CaCl2 ) [23]. The plasmid DNA was extracted using a Miniprep DNA Purification System kit (Promega Corporation, Madison, Wisconsin, USA) following the manufacturer’s protocol. The concentration of recombinant plasmids containing DENV-3 inserts was determined using a Qubit 2.0 fluorometer (Invitrogen, California, USA) with a Qubit dsDNA BR Assay kit (Invitrogen, California, USA) following the manufacturer’s protocol. Serotype-specific DENV-3 primers (DENV-3F GGACTGGACACACGCACTCA, and DENV-3C CATGTCTCTACCTTCTCGACTTGTCT) were used according to Johnson et al. [22], and TaqMan qRT-PCR was also used during the assay. The RT-qPCR was performed using the ABI Prism 7500 Sequence Detection System (Applied Biosystem, California, USA) with a thermalcycling conditions set as follows: a cycle at 50 ◦ C for 2 min, followed by 45 cycles of 95 ◦ C for 10 min, 95 ◦ C for 15 s, and 56 ◦ C for 1 min. The viral load was expressed as number of DENV3 viral particles/mL based on the standard curve constructed using serial dilutions of plasmids containing the DENV-3 insert at concentrations ranging from 8 × 101 to 8 × 107 viral particles/mL.
2.6. Viral load determination by quantitative real time PCR (qPCR) 2.7. Serological analysis For the quantification of viral load, a standard curve was constructed using a plasmid provided by cloning of the amplicon using TOPO TA Cloning Kit (Invitrogen, California, USA),
Measurements of DENV-3 IgM were performed in sera samples by the MAC-ELISA method, according to Kuno et al. [24]. IgM
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129
Hematological Profile A
Leucócitos Totais
Leukogram
D60
D45
D20
D15
D7
D6
D5
D4
D3
D2
D1
D20
D45
D60
D45
D60
D6
D20
D5
D6
D7
D4
D5
D15
D3
D4
D15
D2
D3
D7
D1
D60
D45
D20
D15
↓ D7
D6
D5
D4
D1
D60
D45
D20
D15
D7
D6
D5
D4
0.00
↓
D60
↓
0.75
D3
APTT
1.50
D2
D45
D20
D15
D7
D6
D5
D4
D3
D2
↓ ↓ ↓
↓
D3
0.00
↓
3.00 2.25
D2
↓ ↓
↓
0.75
0.00
1.50
0.75
1.50 0.75
D1
↓
1.50
Hematocrit
D60
D45
D20
D15
D7
D6
D5
D4
D3
D2
2.25
0.00
0.00
↓
D2
↓ ↓
0.75
D1
↓
D1
Platelets D60
PT
Hemoglobin
D45
D20
D7
D15
D6
D5
D4
D3
D2
D1
Red Blood Cells
D45
D20
D7
D6
D5
D4
D3
D2
D1
D60 D60
D45
D20
2.25
1.50
2.25 D1
Neutrophils
D15 D15
D7
D6
D5
D4
D3
D2
D1
↓↓
↓ ↓↓ ↓ ↓ ↓↓ ↓
3.00
↓
Monocytes
↓
0.00
Lymphocytes
0.00
1.50
0.75
1.50 0.75
2.25
↓ ↓
2.25
↓ ↓
0.75
2.25
↓
0.75
1.50
0.00
↓
↓
3.00
↓
↓
1.50
0.00
Hemácia Erythrogram 2.25
↓
2.25
↓
↓
↓↓ ↓
↓
0.75
Coagulogram
C
↓
White Blood Cells
B
↓
1.50
0.00
Baseline fold changes / DENV-3 pre-infecon
↓
2.25
Days aer infecon
Fig. 2. Hematological profile of DENV-3 infected C. penicillata. (A) Leukogram was measured by neutrophil, monocyte, and lymphocyte counts. (B) Erythrogram was measured by red blood cell count, hemoglobin, and hematrocrit determinations. (C) Coagulogram was measured by platelet counts, prothrombin time (PT), and activated partial thromboplastin time (APTT) assays. All data are expressed as baseline fold changes relative to DENV-3 pre-infection. Relevant differences are highlighted by arrows in the figure (↓ = decrease at 1.5 baseline folds).
reactivity was expressed as optical density (OD) at 450 nm. The HIA test was performed as described by Clarke et al. [25], and adapted to microplate by Shope [26], and covered 23 different arboviruses as mentioned in Section 2.3. These data were used to analyze the occurrence of cross-reactivity between the evaluated arboviruses. The results were determined by visual observation of the goose red blood cell sedimentation, and were expressed as reciprocal sera dilution.
2.9. Biochemical studies The renal function was monitored by urea and creatinine (CREA) levels, and the liver function by the aspartate (AST) and alanine (ALT) aminotransferases. The tests were performed using commercial kits (ROCHE, São Paulo, Brazil) in a biochemical analyzer (COBAS MIRA PLUS 400, São Paulo, Brazil), according to the manufacture’s instructions. The results were expressed as IU/mL.
2.8. Hemogram and coagulogram The hemogram was performed in EDTA blood samples using MS4 Hematology Analyzer (Melet Schloesing Laboratoires, France), according to the manufacture’s protocol, and included the following parameters: red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), white blood cells (WBC), neutrophils (NEU), monocytes (MON), lymphocytes (LYM), and platelets (PLT). The coagulogram was performed in sodium citrate plasma samples to measure prothrombin time (PT) and activated partial thromboplastin time (APTT) assays using a CLOTimer coagulometer (Clot, São Paulo, Brazil), as described by the manufacturer.
2.10. Quantification of serum cytokines The level of cytokines (IL-6, TNF-␣, IL-2, IFN-␥, IL-4 and IL-5) was quantified in sera collected from DENV-3 infected marmosets during the experimental study. We used the system Cytometric Bead Array (CBA) (BD Pharmingen, California, USA) according to manufacturer’s protocol. The assay was analyzed using a FACScaliburTM flow cytometer equipped with four color detection system, and the CELLQuestTM software (Becton Dickinson, California, USA) to perform data acquisition. The data analysis was performed using BD CBA software (Becton Dickinson). The results were expressed as mean fluorescence intensity (MFI).
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2.11. Data analysis
Biochemical Profile
3.75 3.00
9.75
9.00 8.25 7.50 6.75
6.00
6.75
6.00
↓
D20
D45
D20
D45
D60
D45 D60 D60
D45
D20
D15
D7
D6
D5
D4
D3
D2
↓ ↓ D1
2.25
1.50 0.75 0.00
↓ ↓
3.00
↓
5.25 4.50 3.75
↓ D15 D20
D2 D3
2.25
1.50 0.75 0.00
D4 D5
3.00
D6 D7
5.25 4.50 3.75
7.50
AST
D60
D7
D6 D6
D15
D5 D5
D15
D4 D4
D7
D2
D3 D3
D1 D1
0.00
↓
D1
Baseline foldchanges / DENV-3pre-infecon
0.75
↓
ALT
1.50
↓
To assess the kinetics of hepatic and renal functions of DENV-3 infected C. penicillata, the serum levels of AST and ALT as well as urea/CREA, respectively, were measured in serum samples along the infection (Fig. 3). Our results showed an early increase in
2.25
↓
Creanine
3.00
↓
3.3. Increased levels of ALT and AST with higher peak at 4 dpi with a persistent decrease of urea along the infection were the major serological markers of DENV-3 infection course in C. penicillata
↓↓
↓ ↓ ↓ ↓↓
↓
Hematological profile of DENV-3 infected C. penicillata was carried out in EDTA whole blood samples. Leukogram, erythrogram and coagulogram data were expressed as baseline fold changes relative to the DENV-3 pre-infection profile. Data analysis demonstrated that despite an early monocytosis at 1 dpi along with a slight neutrophilia at 2 dpi, lymphocytosis at 3 and 60 dpi, and marked decrease in the WBC was observed along the infection at 4, 6, 7, 15, and 45 dpi (Fig. 2A). Our data also demonstrated a decrease in the RBC counts at 3–4 dpi, with clear anemia episodes at 3 and 4 dpi, as demonstrated by lower hemoglobin, and hematocrit values (Fig. 2B). A patent thrombocytopenia was observed along the infection at days 1–15, with sporadic decreases in the APTT values at 2, 6, and 60 dpi (Fig. 2C).
↓
↓
D2
Urea
0.00
3.1. Peak of viremia at 5 dpi along with high anti-DENV-3 IgM reactivity at 15 dpi, and maximum of antibodies levels from 15 to 60 dpi were virological/serological biomarkers in DENV-3 infected C. penicillata
3.2. Despite an early monocytosis at 1 dpi, slight neutrophilia at 2 dpi, and lymphocytosis at 3 dpi, DENV-3 infected C. penicillata developed a persistent leucopenia from 4 to 45 dpi
1.50 0.75
3. Results
Viral load was determinated by RT-qPCR in serum samples of DENV-3 infected C. penicillata throughout the assessment period (1–60 dpi). Our findings demonstrated that virus could be detected as early as 2 dpi with an outstanding peak of viremia at 5 dpi (Fig. 1A). Analysis of the anti-DENV-3 serological reactivity was performed throughout the assessment period (1–60 dpi) by IgM MAC-ELISA as well as by HAI. Our data showed that anti-DENV-3 IgM reached the higher reactivity peak at 15 dpi (Fig. 1B), whereas the maximum of HAI reactivity was achieved from 15 to 60 dpi with a clear cross-reactivity profile amongst DENV serotypes 1–4 (Fig. 1C).
2.25
↓
All data sets, except virological and serological results, were transformed into baseline fold ratio prior to analysis. Baseline fold value was obtained for each parameter as the ratio between the values observed after DENV-3 infection by the baseline value observed at DENV-3 pre-infection. Data analysis was performed considering relevant differences when baseline fold changes were found outside from 0.75 to 1.5 range (gray zones in figures), i.e. relevant decrease when the baseline fold change is lower than 0.75, and relevant increase when the baseline fold change is higher than 1.5. A matrix with all measured biomarkers, expressed as baseline fold changes relative to DENV-3 pre-infection was assembled, and used to calculate the Spearman’s rank correlation indexes. Significant correlations at p < 0.05 indicating interactivity amongst biomarkers were selected to design biomarker networks using open source software (Cytoscape) [27].
↓
130
Days aerinfecon Fig. 3. Biochemical profile of hepatic and renal function of DENV-3 infected C. penicillata. Serum levels of urea, creatinine (CREA), alanine (ALT), and aspartate (AST) aminotransferases are expressed as baseline fold changes relative to DENV-3 preinfection. Relevant differences are highlighted by arrows in the figure (↓ = decrease at 1.5 baseline folds).
the AST levels at 1–4, and 6 dpi with a clear increase in the ALT levels at 2–6 and 7 dpi. Moreover, a persistent decrease in the urea levels was observed with minor changes in the CREA levels (Fig. 3).
3.4. Early increase in TNF-˛ and IFN- levels at 3 dpi with later increases of IFN- at 6, and 15 dpi were found along the DENV-3 infection course in C. penicillata Quantification of serum pro-inflammatory (IL-6, TNF-␣, IL-2, IFN-␥), and type-2 cytokines (IL-4 and IL-5) was carried out (Fig. 4). The results pointed out to increased levels of TNF-␣ and IFN-␥ at 3 dpi, along with later increase of IFN-␥ levels at 6, and 15 dpi (Fig. 4). Isolated decreases in TNF-␣ at 45 dpi, and IL-5 at 7 dpi were also reported (Fig. 4).
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131
Cytokine Profile
D45
D60 D60 D60
D20
D45
D7
D15
D6
D5 D5
D4
D3
D6
D5
D4
D3
D2
↓
D1
D60
D45
D20
D15
D7
D6
D5
D4
0.00
D3
0.00
D2
0.75
D1
0.75
D20
1.50
D45
1.50
D20
2.25
↑
↑
IL-5
2.25
D2
D60
D45
D20
D15
D7
D6
D5
D4
0.00
D3
0.00
D2
0.75
D1
0.75
D1
IL-2
IFN-γ
↑
D15
1.50
D15
1.50
D7
2.25
D7
2.25
D6
D60
D45
D20
D7
D15
D6
D5
D4
D3
D2
0.00
D1
0.00
↓
D4
IL-6
0.75
IL-4
Baseline foldchanges / DENV-3pre-infecon
0.75
D3
1.50
↑
D2
1.50
D1
2.25
TNF-α
2.25
Days aer infecon Fig. 4. Kinetic profile of serum cytokines in DENV-3 infected C. penicillata. Serum cytokines (IL-6, TNF-␣, IL-2, IFN-␥, IL-4, and IL-5) were detected by cytometric bead array (CBA) as described in Section 2. The results are expressed as baseline fold changes relative to DENV-3 pre-infection. Relevant differences are highlighted by arrows in the figure (↓ = decrease at 1.5 baseline folds).
3.5. Two outstanding cytokine network axes (IL-6/IL-2/Viremia/MON/PT, and TNF-˛/LYM/PTL) were reported during early DENV-3 infection in C. penicillata Aiming to assess the interactivity and conections amongst the several biomarkers inclued in our investigation, we have assembled biological networks observed following early and late DENV-3 infection course in C. penicillata (Fig. 5). Our results demonstrated that during early DENV-3 infection, strong correlation axes could be observed in C. penicillata peripheral blood biomarkers, including two major axes. One axes was composed by strong link between IL-6/IL-2/Viremia/MON/PT. Moreover, another axes with strong correlation could be observed between TNF-a/LYM/PTL (Fig. 5 – left network). 3.6. Interactions between IFN-/PLT/DENV-3(IgM;HIA)/PT, and the involvement of type-2 cytokines (IL-4; IL-5) were the major findings of later stages of DENV-3 infection course in C. penicillata The interactivity and connections amongst the biomarkers during the later stages of DENV-3 infection in C. penicillata are shown in Fig. 5. Our data demonstrated that weaker but multiples correlation axes could be identified between C. penicillata peripheral blood biomarkers, including several axes of negative correlation. Interactions between IFN-␥/PLT/DENV-3(IgM;HIA) with negative
correlation with PT could be observed. Furthermore, another axes involving type-2 cytokines (IL-4; IL-5) could also be identified (Fig. 5 – right network).
4. Discussion Dengue is a mosquito-borne viral disease of expanding geographical range and incidence, and is considered the most important and widespread disease caused by arbovirus worldwide, affecting more than 100 countries. Infection by one of the four serotypes of DENV induces a spectrum of disease manifestations, ranging from asymptomatic to life-threatening DHF/DSS. Many efforts have been made to elucidate several aspects of DENVinduced disease, but its pathogenesis is complex and remains unclear. Understanding the mechanisms involved in the early stages of infection is crucial to determine and develop safe therapeutics to prevent the severe outcomes of disease without interfering with control of infection [26]. A common major technical barrier in understanding the pathogenesis of DENV infection is the absence of a suitable animal model that mimics completely dengue disease [28,29]. This fact is justified, for example, by the limited infectivity and modest DENV replication observed in murine models. The purpose of our study was
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Fig. 5. Biomarker networks in DENV-3 infected C. penicillata. Biomarker network were assembled early 1–7 (left panel), and late 15–60 (right panel) following DENV-3 infection according to baseline changes. Node patterns are used to indicate decreased (white dashed), increased (black), oscillating (black dashed) or stable (white) values. Correlations between biomarker pairs are represented by continuous (positive) or dashed (negative) lines. The strength of the correlations is represented by thin line (moderate, r = 0.36–0.67) or thick line (strong, r = 0.68–1.00), according to Taylor [31]. WBC – white blood cells; NEU – neutrophils; MON – monocytes; LYM – lymphocytes; RBC – red blood cells; HCT – hematocrit; HB – hemoglobin; PLT – platelets; PT – prothrombin time; APPT – activated partial thromboplastin time; CREA – creatinine; ALT – alanine aminotransferase; AST – aspartate aminotransferase; VL – viral load; IgM – DENV-3 IgM; HIA – hemmaglutination inhibition assay IgM/IgG.
to develop a feasible experimental model to study dengue virus infection using the C. penicillata non-human primate. Recently, Omatsu et al. [16] performed studies with marmosets of Callithrix jacchus species infected with one of four DENV serotypes isolated from clinical cases of dengue fever in humans, and the establishment of infection was observed after the primary DENV inoculation. The viral RNA was detected in all infected marmosets. Anti-DENV-1, anti-DENV-2, anti-DENV-3 IgM antibodies were detected at 5 dpi, and anti-DENV-4 IgM antibodies were detected at 7 dpi. In this study, a peak of viremia was found at day 5 after DENV-3 infection. Several research studies in humans describe seroepidemiological cross-reactivity between the four serotypes of DENV, with other viruses belonging to Flavivirus genus in DENV infected individuals [30]. In our current study, there was cross-reactivity between the DENV-1, -2 and -4 serotypes. Onlamoon et al. [14] carried out a complete blood cell count in DENV-infected rhesus monkeys. During acute infection, analysis of the erythrogram indicated a slight decrease in the hemoglobin levels, a significant reduction in the hematocrit values, and no changes in the number of RBC. The WBC count showed a significant leukopenia at days 1, 3, 5, 7, 10 after DENV-3 challenge, and leukocytosis at day 14 dpi. Similar to the findings of Onlamoon et al. [14], our study found a slight decrease in hemoglobin and hematocrit values during acute infection caused by DENV-3. Similarly, it was also demonstrated leukopenia caused by a decreased frequency of neutrophils, monocytes, and lymphocytes after DENV-3 infection. Leukopenia may have been caused by a decrease in neutrophil counts, and this alteration is commonly reported in DENV infected individuals, regardless of serotype [32]. Moreover, in our study, we have also observed a slight lymphocytosis at the end of the acute phase, and an early monocytosis at 1 dpi. Our results still showed a modest increase in the levels of AST and ALT, and a significant increase in the APTT during the acute phase. Thrombocytopenia was also observed at 1–15 dpi. Approximately, one-third of DF patients may
have mild hemorrhage manifestations, which may be related to the low platelet count [2]. In this context, thrombocytopenia was an outstanding finding observed in the first days after infection by DENV-3 in marmosets. This finding was similar with those reports found in both humans and rhesus monkeys [14,31]. Indeed, thrombocytopenia was indicative of infection in the experimental model proposed by our study. The immune response after DENV infection is a significant factor associated with disease severity, and induction of symptoms [1]. One of the main components of the immune system against DENV infection includes interferons (IFNs). Gonc¸alves et al. [2] have found that C57BL/6 mice infected with 7.2 × 107 PFU of DENV-1 – strain Mochizuki showed a significant increase of IFN-␥, with peak at 10 dpi. In the current study, we observed a significant increase of IFN-␥ at 3, 6 and 20 dpi. The proinflammatory cytokines seem to play a pathological role during host response to DENV infection. For example, increased levels of TNF-␣ have been associated with severity of dengue manifestation in humans [28]. Similar finding was detected in DENV-3 infected marmosets, which showed a significant increase of TNF-␣ at 3 dpi. The analysis of biomarkers network pointed out two relevant strong axes during early stages of dengue fever, a protective axes TNF-␣/Lymphocytes/Platelets, and a pathological IL-2/IL6/Viremia/Monocyte/PT bond. Later on, the biomarker network highlighted the interaction IFN-␥/PLT/DENV-3(IgM;HIA)/PT, and the involvement of type-2 cytokines (IL-4; IL-5). 5. Conclusions Our data demonstrated that C. penicillata is a feasible experimental model for DENV infection, because it was able to reproduce many aspects of DENV disease in terms of viremia, serological profile, liver damage, relevant alterations in hematological, biochemical, and immunological parameters. Therefore, this model
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could be useful to pathogenesis studies, discovery of novel antiviral drugs as well as to evaluate vaccine candidates against DENV. Competing interests The author(s) declare that they have no competing interests. Acknowledgments This study was supported by grants from Fundac¸ão Oswaldo Cruz (FIOCRUZ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundac¸ão de Amparo à Pesquisa de Minas Gerais (FAPEMIG), and Instituto Nacional de Ciência e Tecnologia para Febres Hemorrágicas Virais (INCT-FHV grant CNPq/CAPES/FAPESPA 573739/2008-0). The authors thank the program for technological development in tools for health-PDTISFIOCRUZ for the use of its facilities. ATC, OAMF, and PFCV thank CNPq for fellowships (PQ); MSF was supported by a fellowship of CAPES. MSF, PHGC, GAS, SMMC, AGDJ, SGR, RSSA, and PFCV designed the study, and performed the experiments; MFCS, DAGZ, SRB, ATC, and OAMF wrote the paper. All authors read and approved the final manuscript. References [1] Herrero LJ, Zakhary A, Gahan ME, Nelson MA, Herring BL, Hapel AJ, et al. Dengue virus therapeutic intervention strategies based on viral, vector and host factors involved in disease pathogenesis. Pharmacol Ther 2013;137: 266–82. [2] Gonc¸alves D, de Queiroz Prado R, Almeida Xavier E, Cristina de Oliveira N, da Matta Guedes PM, da Silva JS, et al. Imunocompetent mice model for dengue virus infection. SciWorld J 2012;2012:1–12. [3] Yacoub S, Mongkolsapaya J, Screaton G. The pathogenesis of dengue. Curr Opin Infect Dis 2013;29:7221–8. [4] Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, et al. The global distribution and burden of dengue. Nature 2013;25:504–7. [5] Gubler DJ. Dengue and dengue hemorrhagic fever: its history and resurgence as a global public health problem. In: Gubler DJ, Kuno G, editors. Dengue and dengue hemorrhagic fever. London: Cab International; 1997. [6] Murray PR, Rosenthal KS, Kobayashi GS, Pfaller MA. Microbiologia médica. 4th ed. Rio de Janeiro: Guanabara Koogan; 2000. [7] Guzman MG, Alvarez M, Halstead SB. Secondary infection as a risk factor for dengue hemorrhagic fever/dengue shock syndrome: an historical perspective and role of antibody-dependent enhancement of infection. Arch Virol 2013:1–15. [8] BRASIL. Ministério da Saúde Guia de vigilância epidemiológica. Brasília: Fundac¸ão Nacional de Saúde; 2002. [9] Chen WF, Yeh WT, Yang MY. A model of the real-time correlation of viral titers with immune reactions in antibody-dependent enhancement of dengue2 infection. FEMS Immunol Med Microbiol 2001;30:1–7. [10] Kuruvilla JG, Troyer RM, Devi S, Akkina R. Dengue virus infection and immune response in humanized RAG2(−/−)gamma(c)(−/−) (RAG-hu) mice. Virology 2007;369:143–52.
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