Isolation of dengue virus serotype 4 genotype II from a patient with high viral load and a mixed Th1/Th17 inflammatory cytokine profile in South Brazil
- Diogo Kuczera†1,
- Lorena Bavia†1,
- Ana Luiza Pamplona Mosimann1,
- Andrea Cristine Koishi1,
- Giovanny Augusto Camacho Antevere Mazzarotto1,
- Mateus Nóbrega Aoki1,
- Ana Maria Ferrari Mansano2,
- Ediléia Inês Tomeleri2,
- Wilson Liuti Costa Junior2,
- Milena Menegazzo Miranda3,
- Maria Lo Sarzi2,
- Wander Rogério Pavanelli3,
- Ivete Conchon-Costa3,
- Claudia Nunes Duarte dos Santos1Email author and
- Juliano Bordignon1Email author
© The Author(s). 2016
Received: 21 January 2016
Accepted: 24 May 2016
Published: 6 June 2016
We report the isolation and characterization of dengue virus (DENV) serotype 4 from a resident of Santa Fé, state of Paraná, South Brazil, in March 2013. This patient presented with hemorrhagic manifestations, high viral load and, interestingly, a mixed Th1/Th17 cytokine profile.
The patient presented with classical dengue symptoms, such as fever, rash, myalgia, arthralgia, and hemorrhagic manifestations including petechiae, gum bleeding and a positive tourniquet test result. A serum sample obtained 1 day after the initial appearance of clinical symptoms was positive for NS1 viral antigen, but this sample was negative for both IgM and IgG against DENV. Dengue virus infection was confirmed by isolation of the virus from C6/36 cells, and dengue virus serotyping was performed via one-step RT-PCR. The infection was confirmed to be caused by a serotype 4 dengue virus. Additionally, based on multiple alignment and phylogeny analyses of its complete genome sequence, the viral strain was classified as genotype II (termed LRV13/422). Moreover, a mixed Th1/Th17 cytokine profile was detected in the patient’s serum, and this result demonstrated significant inflammation. Biological characterization of the virus via in vitro assays comparing LRV13/422 with a laboratory-adapted reference strain of dengue virus serotype 4 (TVP/360) showed that LRV13/422 infects both vertebrate and invertebrate cell lines more efficiently than TVP/360. However, LRV13/422 was unable to inhibit type I interferon responses, as suggested by the results obtained for other dengue virus strains. Furthermore, LRV13/422 is the first completely sequenced serotype 4 dengue virus isolated in South Brazil.
The high viral load and mixed Th1/Th17 cytokine profile observed in the patient’s serum could have implications for the development of the hemorrhagic signs observed, and these potential relationships can now be further studied using suitable animal models and/or in vitro systems.
Dengue virus serotype 4 (DENV-4) genotype II first circulated in Brazil in 1981 and 1982 in limited outbreaks in Boa Vista (Roraima), North Brazil , though no other case related to this serotype was reported in the next 25 years. The resurgence of DENV-4 was reported in Manaus (the Amazon region of Brazil) in 2008, when the virus was isolated from serum samples of three patients with no recent travel history . Phylogenetic analyses indicated that these viruses belong to genotype I, which were circulating in Asian countries but not in the Americas . In 2010, the reemergence of DENV-4 was officially recognized by the Brazilian Ministry of Health when it was again notified cases in the northern region of Brazil (Boa Vista, Roraima) . The strains isolated at that time were classified as genotype II . Currently, dengue virus in Brazil is considered endemic, and four dengue virus serotypes are co-circulating . Here, we describe a non-fatal clinical case of dengue virus from a patient with high viral load and a mixed Th1/Th17 cytokine profile. Additionally, phylogenetic analyses and in vitro biological characterization of the new virus strain were performed.
This study addressed a 45-year-old Caucasian man who was a resident of the county of Santa Fé (23° 2′ 16″ S, 51° 48′ 18″ W), located at the North region of the state of Paraná, in South Brazil. The patient presented the first signs and symptoms of dengue virus infection at Cambé (23° 16′ 33″ S, 51° 16′ 40″ W), a city 87.1 km from Santa Fé (Additional file 1: Figure S1). The patient presented at a public health system unit on March 20, 2013, 1 day after the beginning of symptoms, which included fever, rash, myalgia, arthralgia, diarrhea, prostration, itch, retro-orbital pain, nausea, petechiae and gum bleeding. Additionally, the tourniquet test assessing capillary fragility produced positive results. A peripheral blood sample was collected after the patient’s consent with the approval of the FIOCRUZ Research Ethics Committee (n°. 617/11). The patient’s serum tested positive for NS1 antigen (PanBio, QLD, Australia) but negative for antibodies IgM (PanBio, QLD, Australia) and IgG (PanBio, QLD, Australia) against dengue virus.
Virus isolation and serotyping
Primer sequences used for each dengue virus serotype detection
Primer sequence (5′-3′)
Genome position (GenBank accession number)
132-159 (NC_001477 e NC_001475)
Patient viremia and serum cytokine profile
In addition to the viral load, the T helper response plays a role in the manifestation of hemorrhage in association with dengue virus infection . Because the patient presented with hemorrhagic symptoms (petechiae and gum bleeding), thrombocytopenia and a high viral load, we decided to evaluate the T helper cytokine profile using a human Th1/Th2/Th17 BD™ Cytometric Bead Array kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s recommendations. The data demonstrated low levels of IL-2 and the secretion of IL-4, IL-10 and TNF-α. However, the presence of high levels of IFN-γ, IL-17A and IL-6 (compared to the levels of these cytokines in adult healthy donors)  suggested that a mixed Th1/Th17 cytokine response could play a role in both dengue virus replication control and hemorrhagic manifestations (Fig. 2c). A Th1-type response has been observed more frequently in dengue fever (DF) patients than in patients with severe dengue virus infection . Additionally, IFN-γ, a Th1 cytokine, was essential for DENV replication control and host resistance in a mouse model, and these results indicated a protective role of this cytokine during dengue virus infection . However, it was shown that high levels of cytokines produced by T cells, macrophages/monocytes and endothelial cells (TNF-α, IFN-γ, IL-10, IL-6 and IL-8) contributed to endothelial cell damage and hemorrhagic manifestations due to a phenomenon known as a cytokine storm [14–16]. Additionally, in dengue-infected patients, the IFN-γ levels were increased in severe cases compared to cases of mild disease . Moreover, treatment with anti-TNF-α antibodies significantly reduced mortality in a mouse model of lethal dengue virus serotype 2 infection, thus reinforcing the role of cytokines in dengue pathogenesis .
Furthermore, the role of IL-17 in the severity of dengue fever has not been well elucidated. Arias and colleagues showed that an increase in IL-17 expression was not associated with the severity of disease, primary or secondary infection or DENV serotype . On the other hand, Jain and colleagues demonstrated that high levels of IL-17 were associated with severe dengue infection compared to dengue without warning signs . Despite the observation of a high level of IL-17 in the patient’s serum, the effect of IL-17 on disease severity must be evaluated using a larger number of samples before concluding that this cytokine plays a protective or pathogenic role in dengue virus infection. Additionally, many pro-inflammatory mediators are produced in response to IL-17, such as IL-6 and neutrophil- and granulocyte-attracting chemokines, and the levels of these factors are increased during DENV infection [20, 21].
High levels of IL-6 have been related to dengue pathogenesis and hemorrhagic symptoms and have been established as a biomarker of DENV infection [15, 22, 23]. This cytokine mediates the increases in endothelial cell permeability and in the production of anti-platelet or anti-endothelial cell autoantibodies, leading to plasma leakage and bleeding [23, 24].
In this fashion, a mixed cytokine profile (Th1/Th17) might induce an important and protective (controlling virus replication) pro-inflammatory response. However, a function of secreted cytokines in promoting the development of the hemorrhagic manifestations observed in the patient cannot be ruled out [14–16].
In vitro characterization
Sequencing and phylogenetic analyses
Since the reemergence of DENV-4 in 2010 in Brazil, co-circulation of four dengue virus serotypes has occurred. Additionally, the hyperendemicity of dengue virus observed in Brazil enhanced the severity of the disease and the mortality rate of infection. The isolation of a DENV-4 genotype II in South Brazil five years after entrance of the virus into the northern region of the country is relevant to understanding the spread and epidemiology of dengue virus. Moreover, isolation of the LRV13/422 strain will be useful for studying host-pathogen interactions, diagnosis, immune responses and antiviral development.
°C, celsius; μL, microliter; DENV, dengue virus; DF, dengue fever; dpi, days post-infection; E, envelope; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FFU, focus-forming units; h, hour(s); hpi, hours post-infection; IFA, immunofluorescence assay; IFN, interferon; IgG, immunoglobulin G; IgM, immunoglobulin M; IL, interleukin; IU, international unit; MOI, multiplicity of infection; NS1, non-structural protein 1; PBS, phosphate-buffered saline; RT-PCR, reverse-transcription polymerase chain reaction; S, South; Th, T helper; TNF, tumor necrosis factor; v/v, volume/volume; W, West
The authors thank the Program for Technological Development in Tools for Health-PDTIS-FIOCRUZ for use of its facilities (RPT07C; Microscopy Facility and RPT08 L; Flow Cytometry Facility at the Carlos Chagas Institute/Fiocruz-PR, Brazil). The authors also thank Bruna Hilzendeger Marcon for assistance with microscopic analysis, as well as Dra. Pryscilla F. Wowk (ICC/Fiocruz) and Dra. Alessandra Abel Borges (UFAL) for critically reading the manuscript. The authors thank Itamar Crispim for producing Additional file 1: Figure S1. The authors thank CNPq PROCAD/Casadinho, PAPES/Fiocruz, Brazilian Ministry of Health (PPSUS-2012)/Fundação Araucária, for providing financial support.
DK Manuscript writing, virus detection isolation and RT-PCR. MNA Serology assays. MMM, MS, AMFM, EIT, WLCJ Sample and clinical data collection. LB Manuscript writing, figures, in vitro assays, and statistical analysis. ACK Interferon assay and in vitro assays. ALPM Nucleotide sequencing and phylogenetic analysis. GACAM In vitro assays. WRP, ICC, CNDS, JB Study conception and design and manuscript writing. DK and LB contributed equally to this work. All authors read and approved the final manuscript.
The authors declare that they have no competing interest.
Consent for publication
The patient provided written consent for participation in the study and for publication of the results.
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