Recently differentiated epimastigotes from Trypanosoma cruzi are infective to the mammalian host.

Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle in which four distinct developmental forms alternate between the insect vector and the mammalian host. It is assumed that replicating epimastigotes present in the insect gut are not infective to mammalian host, a paradigm corroborated by the widely acknowledged fact that only this stage is susceptible to the complement system. In the present work, we establish a T. cruzi in vitro and in vivo epimastigogenesis model to analyze the biological aspects of recently differentiated epimastigotes (rdEpi). We show that both trypomastigote stages of T. cruzi (cell-derived and metacyclic) are able to transform into epimastigotes (processes termed primary and secondary epimastigogenesis, respectively) and that rdEpi have striking properties in comparison to long-term cultured epimastigotes: resistance to complement-mediated lysis and both in vitro (cell culture) and in vivo (mouse) infectivity. Proteomics analysis of all T. cruzi stages reveled a cluster of proteins that were up-regulated only in rdEpi (including ABC transporters and ERO1), suggesting a role for them in rdEpi virulence. The present work introduces a new experimental model for the study of host-parasite interactions, showing that rdEpi can be infective to the mammalian host.

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. CASE PRESENTATION: 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. CONCLUSION: 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.

Development, characterization and application of monoclonal antibodies against Brazilian Dengue virus isolates.

Dengue is the most prevalent human arboviral disease. The morbidity related to dengue infection supports the need for an early, quick and effective diagnostic test. Brazil is a hotspot for dengue, but no serological diagnostic test has been produced using Brazilian dengue virus isolates. This study aims to improve the development of immunodiagnostic methods for dengue virus (DENV) detection through the production and characterization of 22 monoclonal antibodies (mAbs) against Brazilian isolates of DENV-1, -2 and -3. The mAbs include IgG2bκ, IgG2aκ and IgG1κ isotypes, and most were raised against the envelope or the pre-membrane proteins of DENV. When the antibodies were tested against the four DENV serotypes, different reactivity patterns were identified: group-specific, subcomplex specific (DENV-1, -3 and -4 and DENV-2 and -3) and dengue serotype-specific (DENV-2 or -3). Additionally, some mAbs cross-reacted with yellow fever virus (YFV), West Nile virus (WNV) and Saint Louis encephalitis virus (SLEV). None of the mAbs recognized the alphavirus Venezuelan equine encephalitis virus (VEEV). Furthermore, mAbs D3 424/8G, D1 606/A12/B9 and D1 695/12C/2H were used to develop a capture enzyme-linked immunosorbent assay (ELISA) for anti-dengue IgM detection in sera from patients with acute dengue. To our knowledge, these are the first monoclonal antibodies raised against Brazilian DENV isolates, and they may be of special interest in the development of diagnostic assays, as well as for basic research.