Intrahost genetic diversity is thought to facilitate arbovirus adaptation to changing environments and hosts, and it might also be linked to viral pathogenesis. Dengue virus serotype 2 (DENV-2) has circulated in Brazil since 1990 and is associated with severe disease and explosive outbreaks. Intending to shed light on the viral determinants for severe dengue pathogenesis, we sought to analyze the DENV-2 intrahost genetic diversity in 68 patient cases clinically classified as dengue fever (n = 31), dengue with warning signs (n = 19), and severe dengue (n = 18). Unlike previous DENV intrahost diversity studies whose approaches employed PCR, here we performed viral whole-genome deep sequencing from clinical samples with an amplicon-free approach, representing the real intrahost diversity scenario. Striking differences were detected in the viral population structure between the three clinical categories, which appear to be driven mainly by different infection times and selection pressures, rather than being linked with the clinical outcome itself. Diversity in the NS2B gene, however, showed to be constrained, irrespective of clinical outcome and infection time. Finally, 385 non-synonymous intrahost single-nucleotide variants located along the viral polyprotein, plus variants located in the untranslated regions, were consistently identified among the samples. Of them, 124 were exclusively or highly detected among cases with warning signs and among severe cases. However, there was no variant that by itself appeared to characterize the cases of greater severity, either due to its low intrahost frequency or the conservative effect on amino acid substitution. Although further studies are necessary to determine their real effect on viral proteins, this heightens the possibility of epistatic interactions. The present analysis represents an initial effort to correlate DENV-2 genetic diversity to its pathogenic potential and thus contribute to understanding the virus’s dynamics within its human host.

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ABSTRACT. This study describes the development of equipment capable of transporting decellularization fluid through an organ by perfusion to create an in vitro acellular scaffold that maintains the three-dimensional architecture of the organ. The equipment was designed to be compatible with several existing decellularization protocols and can be used to modify the flow rate, perfusion pressure, and temperature in each decellularization protocol as well as the technique of cannulation of the organ in a controlled manner. The device was tested with chicken hearts and efficiently accomplished decellularization using the perfusion method. Further amendments can be made to improve the efficiency of the decellularization of diverse organs. It is expected that this equipment will aid in the characterization and improvement of the decellularization process and may have future applications in the process of recellularization of different organs.Keywords: decellularization; cardiac bioengineering; biomaterials; perfusion process; heart decellularization Desenvolvimento de equipamento para decelularização pelo método de perfusão RESUMO. O presente estudo descreve o desenvolvimento de um equipamento capaz de transportar o fluido de decelularização através do órgão inteiro, por perfusão, para criar uma estrutura acelular in vitro que mantém a arquitetura tridimensional completa do órgão. O equipamento foi concebido ser compatível com vários protocolos de decelularização existentes e pode ser utilizado para modificar a vazão, a pressão e a temperatura de perfusão em cada protocolo, bem como a técnica de canulação do órgão de uma forma controlada. O dispositivo foi testado com corações de galinha e executou eficientemente o protocolo de decelularização, utilizando o método de perfusão. Outras alterações podem ser feitas para melhorar a eficiência da decelularização em diversos órgãos. Espera-se que este equipamento auxilie na caracterização e melhoria do processo de decelularização além de possibilitar futuras aplicações no processo de recelularização de diferentes órgãos.Palavras-chave: decelularização; bioengenharia cardíaca; biomateriais; processo de perfusão; decelularização de coração.