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.
BACKGROUND
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.


METHODS
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.


CONCLUSIONS
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.
Laboratório de Virologia Molecular, Instituto Carlos Chagas, Fiocruz, Curitiba, PR, Brazil.
Diogo
D
Kuczera
Laboratório de Virologia Molecular, Instituto Carlos Chagas, Fiocruz, Curitiba, PR, Brazil.
Lorena
L
Bavia
Laboratório de Virologia Molecular, Instituto Carlos Chagas, Fiocruz, Curitiba, PR, Brazil.
Ana Luiza Pamplona
AL
Mosimann
Laboratório de Virologia Molecular, Instituto Carlos Chagas, Fiocruz, Curitiba, PR, Brazil.
Andrea Cristine
AC
Koishi
Laboratório de Virologia Molecular, Instituto Carlos Chagas, Fiocruz, Curitiba, PR, Brazil.
Giovanny Augusto Camacho Antevere
GA
Mazzarotto
Laboratório de Virologia Molecular, Instituto Carlos Chagas, Fiocruz, Curitiba, PR, Brazil.
Mateus Nóbrega
MN
Aoki
Secretaria Municipal de Saúde de Cambé, Cambé, PR, Brazil.
Ana Maria Ferrari
AM
Mansano
Secretaria Municipal de Saúde de Cambé, Cambé, PR, Brazil.
Ediléia Inês
EI
Tomeleri
Secretaria Municipal de Saúde de Cambé, Cambé, PR, Brazil.
Wilson Liuti
WL
Costa Junior
Laboratório de Imunoparasitologia Experimental, Universidade Estadual de Londrina, Londrina, PR, Brazil.
Milena Menegazzo
MM
Miranda
Secretaria Municipal de Saúde de Cambé, Cambé, PR, Brazil.
Maria
M
Lo Sarzi
Laboratório de Imunoparasitologia Experimental, Universidade Estadual de Londrina, Londrina, PR, Brazil.
Wander Rogério
WR
Pavanelli
Laboratório de Imunoparasitologia Experimental, Universidade Estadual de Londrina, Londrina, PR, Brazil.
Ivete
I
Conchon-Costa
Laboratório de Virologia Molecular, Instituto Carlos Chagas, Fiocruz, Curitiba, PR, Brazil. clsantos@fiocruz.br.
Claudia Nunes
CN
Duarte Dos Santos
Laboratório de Virologia Molecular, Instituto Carlos Chagas, Fiocruz, Curitiba, PR, Brazil. julianobordignon@fiocruz.br.
Juliano
J
Bordignon
D000818:Animals; D001938:Brazil; D002460:Cell Line; D016000:Cluster Analysis; D016207:Cytokines / Q000097:blood*; D003715:Dengue / Q000473:pathology* / Q000821:virology*; D003716:Dengue Virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005838:Genotype; D006801:Humans; D007448:Invertebrates; D008297:Male; D008875:Middle Aged; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D065288:Serogroup; D018417:Th1 Cells / Q000276:immunology; D058504:Th17 Cells / Q000276:immunology; D014714:Vertebrates; D019562:Viral Load; D014776:Virus Cultivation
D016207:Cytokines
Cytokines; Dengue virus serotype 4; Genetic characterization; IFN-gamma; IL-17; IL-6; Paraná State; Viremia