Benznidazole (BZ) is the most commonly used drug for the treatment of Chagas disease. Although BZ is known to induce the formation of free radicals and electrophilic metabolites within the parasite Trypanosoma cruzi, its precise mechanisms of action are still elusive. Here, we analyzed the survival of T. cruzi exposed to BZ using genetically modified parasites overexpressing different DNA repair proteins. Our results indicate that BZ induces oxidation mainly in the nucleotide pool, as heterologous expression of the nucleotide pyrophosphohydrolase MutT (but not overexpression of the glycosylase TcOgg1) increased drug resistance in the parasite. In addition, electron microscopy indicated that BZ catalyzes the formation of double-stranded breaks in the parasite, as its genomic DNA undergoes extensive heterochromatin unpacking following exposure to the drug. Furthermore, the overexpression of proteins involved in the recombination-mediated DNA repair increased resistance to BZ, reinforcing the idea that the drug causes double-stranded breaks. Our results also show that the overexpression of mitochondrial DNA repair proteins increase parasite survival upon BZ exposure, indicating that the drug induces lesions in the mitochondrial DNA as well. These findings suggest that BZ preferentially oxidizes the nucleotide pool, and the extensive incorporation of oxidized nucleotides during DNA replication leads to potentially lethal double-stranded DNA breaks in T. cruzi DNA.

SummaryBackgroundZika virus infections and suspected microcephaly cases have been reported in Angola since late 2016, but no data are available about the origins, epidemiology, and diversity of the virus. We aimed to investigate the emergence and circulation of Zika virus in Angola.MethodsDiagnostic samples collected by the Angolan Ministry of Health as part of routine arboviral surveillance were tested by real-time reverse transcription PCR by the Instituto Nacional de Investigação em Saúde (Ministry of Health, Luanda, Angola). To identify further samples positive for Zika virus and appropriate for genomic sequencing, we also tested samples from a 2017 study of people with HIV in Luanda. Portable sequencing was used to generate Angolan Zika virus genome sequences from three people positive for Zika virus infection by real-time reverse transcription PCR, including one neonate with microcephaly. Genetic and mobility data were analysed to investigate the date of introduction and geographical origin of Zika virus in Angola. Brain CT and MRI, and serological assays were done on a child with microcephaly to confirm microcephaly and assess previous Zika virus infection.FindingsSerum samples from 54 people with suspected acute Zika virus infection, 76 infants with suspected microcephaly, 24 mothers of infants with suspected microcephaly, 336 patients with suspected dengue virus or chikungunya virus infection, and 349 samples from the HIV study were tested by real-time reverse transcription PCR. Four cases identified between December, 2016, and June, 2017, tested positive for Zika virus. Analyses of viral genomic and human mobility data suggest that Zika virus was probably introduced to Angola from Brazil between July, 2015, and June, 2016. This introduction probably initiated local circulation of Zika virus in Angola that continued until at least June, 2017. The infant with microcephaly in whom CT and MRI were done had brain abnormalities consistent with congenital Zika syndrome and serological evidence for Zika virus infection.InterpretationOur analyses show that autochthonous transmission of the Asian lineage of Zika virus has taken place in Africa. Zika virus surveillance and surveillance of associated cases of microcephaly throughout the continent is crucial.FundingRoyal Society, Wellcome Trust, Global Challenges Research Fund (UK Research and Innovation), Africa Oxford, John Fell Fund, Oxford Martin School, European Research Council, Departamento de Ciência e Tecnologia/Ministério da Saúde/National Council for Scientific and Technological Development, and Ministério da Educação/Coordenação de Aperfeicoamento de Pessoal de Nível Superior.

Zika virus (ZIKV) infection during pregnancy can cause a set of severe abnormalities in the fetus known as congenital Zika syndrome (CZS). Experiments with animal models and in vitro systems have substantially contributed to our understanding of the pathophysiology of ZIKV infection. Here, to investigate the molecular basis of CZS in humans, we used a systems biology approach to integrate transcriptomic, proteomic, and genomic data from the postmortem brains of neonates with CZS. We observed that collagens were greatly reduced in expression in CZS brains at both the RNA and protein levels and that neonates with CZS had several single-nucleotide polymorphisms in collagen-encoding genes that are associated with osteogenesis imperfecta and arthrogryposis. These findings were validated by immunohistochemistry and comparative analysis of collagen abundance in ZIKV-infected and uninfected samples. In addition, we showed a ZIKV-dependent increase in the expression of cell adhesion factors that are essential for neurite outgrowth and axon guidance, findings that are consistent with the neuronal migration defects observed in CZS. Together, these findings provide insights into the underlying molecular alterations in the ZIKV-infected brain and reveal host genes associated with CZS susceptibility.

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Specific DNA repair pathways from Trypanosoma cruzi are believed to protect genomic DNA and kinetoplast DNA (kDNA) from mutations. Particular pathways are supposed to operate in order to repair nucleotides oxidized by reactive oxygen species (ROS) during parasite infection, being 7,8-dihydro-8-oxoguanine (8oxoG) a frequent and highly mutagenic base alteration. If unrepaired, 8oxoG can lead to cytotoxic base transversions during DNA replication. In mammals, DNA polymerase beta (Polβ) is mainly involved in base excision repair (BER) of oxidative damage. However its biological role in T. cruzi is still unknown. We show, by immunofluorescence localization, that T. cruzi DNA polymerase beta (Tcpolβ) is restricted to the antipodal sites of kDNA in replicative epimastigote and amastigote developmental stages, being strictly localized to kDNA antipodal sites between G1/S and early G2 phase in replicative epimastigotes. Nevertheless, this polymerase was detected inside the mitochondrial matrix of trypomastigote forms, which are not able to replicate in culture. Parasites over expressing Tcpolβ showed reduced levels of 8oxoG in kDNA and an increased survival after treatment with hydrogen peroxide when compared to control cells. However, this resistance was lost after treating Tcpolβ overexpressors with methoxiamine, a potent BER inhibitor. Curiously, a presumed DNA repair focus containing Tcpolβ was identified in the vicinity of kDNA of cultured wild type epimastigotes after treatment with hydrogen peroxide. Taken together our data suggest participation of Tcpolβ during kDNA replication and repair of oxidative DNA damage induced by genotoxic stress in this organelle.