Phenotypic and Genetic Studies of the Viral Lineage Associated with the Recent Yellow Fever Outbreak in Brazil.

Yellow fever virus (YFV) caused an outbreak in the Brazilian Southeast from 2016 to 2019, of the most significant magnitude since the 1900s. An investigation of the circulating virus revealed that most of the genomes detected in this period carried nine unique amino acid polymorphisms, with eight located in the non-structural proteins NS3 and NS5, which are pivotal for viral replication. To elucidate the effect of these amino acid changes on viral infection, we constructed viruses carrying amino acid alterations in NS3 and NS5, performed infection in different cells, and assessed their neurovirulence in BALB/c mice and infected AG129 mice. We observed that the residues that compose the YFV 2016-2019 molecular signature in the NS5 protein might have been related to an attenuated phenotype, and that the alterations in the NS3 protein only slightly affected viral infection in AG129 mice, increasing to a low extent the mortality rate of these animals. These results contributed to unveiling the role of specific naturally occurring amino acid changes in the circulating strain of YFV in Brazil.

Phenotypic and Genetic Variability of Isolates of ZIKV-2016 in Brazil.

The possibility of a Zika virus epidemic resurgence requires studies to understand its mechanisms of pathogenicity. Here, we describe the isolation of the Zika virus from breast milk (Rio-BM1) and compare its genetic and virological properties with two other isolates (Rio-U1 and Rio-S1) obtained during the same epidemic period. Complete genomic analysis of these three viral isolates showed that they carry characteristics of the American isolates and belong to the Asian genotype. Furthermore, we detected eight non-synonymous single nucleotide variants and multiple nucleotide polymorphisms that reflect phenotypic changes. The new isolate, Rio-BM1, showed the lowest replication rates in mammalian cells, induced lower cell death rates, was more susceptible to treatment with type I IFN, and was less pathogenic than Rio-U1 in a murine model. In conclusion, the present study shows evidence that the isolate Rio-BM1 is more attenuated than Rio-U1, probably due to the impact of genetic alterations in the modulation of virulence. The results obtained in our in vitro model were consistent with the pathogenicity observed in the animal model, indicating that this method can be used to assess the virulence level of other isolates or to predict the pathogenicity of reverse genetic constructs containing other polymorphisms.

Biological Characterization of Yellow Fever Viruses Isolated From Non-human Primates in Brazil With Distinct Genomic Landscapes.

Since the beginning of the XXI Century, the yellow fever virus (YFV) has been cyclically spreading from the Amazon basin to Brazil's South and Southeast regions, culminating in an unprecedented outbreak that started in 2016. In this work, we studied four YFV isolated from non-human primates obtained during outbreaks in the states of Rio Grande do Sul in 2008 (PR4408), Goiás (GO05), and Espírito Santo (ES-504) in 2017, and Rio de Janeiro (RJ 155) in 2019. These isolates have genomic differences mainly distributed in non-structural proteins. We compared the isolates' rates of infection in mammal and mosquito cells and neurovirulence in adult mice. RJ 155 and PR4408 YFV isolates exhibited higher infectivity in mammalian cells and neurovirulence in mice. In mosquito Aag2 cells, GO05 and PR4408 displayed the lowest proliferation rates. These results suggest that RJ 155 and PR4408 YFV isolates carry some genomic markers that increase infectivity in mammal hosts. From this characterization, it is possible to contribute to discovering new molecular markers for the virulence of YFV.

Recovery of Synthetic Zika Virus Based on Rio-U1 Isolate Using a Genetically Stable Two Plasmid System and cDNA Amplification.

In 2016, the world experienced the unprecedented Zika epidemic. The ZIKV emerged as a major human pathogen due to its association with the impairment of perinatal development and Guillain-Barré syndrome. The occurrence of these severe cases of Zika points to the significance of studies for understanding the molecular determinants of flavivirus pathogenesis. Reverse genetics is a powerful method for studying the replication and determinants of pathogenesis, virulence, and viral attenuation of flaviviruses, facilitating the design of vaccines and therapeutics. However, the main hurdle in the development of infectious clones is the instability of full-length cDNA in Escherichia coli. Here, we described the development of a genetically stable and efficient infectious clone based on the ZIKV Rio-U1 isolated in the 2016 epidemic in Brazil. The employed strategy consisted of cloning the viral cDNA genome into two stable plasmid subclones and obtaining a high-quality cDNA template with increment in DNA mass for in vitro transcription by PCR amplification. The strategy for developing a ZIKV infectious cDNA clone designed in this study was successful, yielding a replicative and efficient clone-derived virus with high similarities with its parental virus, Rio-U1, by comparison of the proliferation capacity in mammal and insect cells. The infection of AG129 immunocompromised mice caused identical mortality rates, with similar disease progression and morbidity in the animals infected with the parental and the cDNA-derived virus. Histopathological analyses of mouse brains infected with the parental and the cDNA-derived viruses revealed a similar pathogenesis degree. We observed meningoencephalitis, cellular pyknosis, and neutrophilic invasion adjacent to the choroid plexus and perivascular cuffs with the presence of neutrophils. The developed infectious clone will be a tool for genetic and functional studies in vitro and in vivo to understand viral infection and pathogenesis better.

Obtenção de clones infecciosos de ZIKV para estudos de atenuação viral

O vírus Zika (ZIKV) é um importante patógeno humano, associado a complicações graves como a síndrome congênita do ZIKV em neonatos, e a síndrome de Guillain-Barré em adultos. Até o momento, não estão disponíveis vacinas ou drogas antivirais contra a infecção por ZIKV, tornando-se um importante desafio o seu desenvolvimento. Esse trabalho teve como objetivo a construção de variantes de ZIKV atenuadas, obtidas pela tecnologia de genética reversa envolvendo plasmídeos bacterianos. Conhecidamente, o cDNA de ZIKV é instável em Escherichia coli, assim, foi estabelecida uma metodologia de construção de ZIKV sintético, baseado no vírus epidêmico Rio-U1, através clonagem do cDNA de ZIKV em um sistema de dois plasmídeos, utilizando o pCC1, um plasmídeo de cópia única. Para mitigar as restrições de rendimento de massa de cDNA, acoplou-se reações de PCR em diferentes etapas do protocolo, favorecendo a montagem do molde completo de cDNA de ZIKV, possibilitando a recuperação viral após transcrição in vitro. O clone IC.ZIKV-Rio-U1 demonstrou padrões de proliferação viral em modelos celulares muito similares ao vírus parental. Além disso, se mostrou virulento em camundongos AG129, exibindo os mesmos sinais de acometimento neurológico desenvolvidos pelo vírus parental. A partir da obtenção de IC.ZIKV-Rio-U1, foram desenvolvidas duas abordagens para promoção de atenuação viral, ambas envolvendo os genes NS4B e NS5. A primeira, foi baseada no fato de que, ao comparar ZIKV circulantes nas Américas com a linhagem ancestral Malásia de 1966, foi observado que a maioria das mutações sinônimas e não sinônimas ocorreu nas proteínas NS4B e NS5. Para avaliar se essas mutações influenciaram na virulência de ZIKV, foi construído o vírus quimérico IC.ZIKV-ns4b-ns5/Mal, a partir da substituição dos genes NS4B e NS5 de ZIKV Rio-U1 pelos genes equivalentes, derivados da linhagem ancestral. Não houve diferença significativa na virulência dos vírus IC.ZIKV-ns4b-ns5/Mal e IC.ZIKV-Rio-U1 em diferentes modelos celulares, mas foi observada uma ligeira diminuição na virulência em modelo de camundongos AG129. A segunda abordagem consistiu na desotimização de pares de códons dos genes NS4B e NS5, através de permuta nucleotídica baseada em dois algoritmos distintos, dN231 e N3, criando os vírus IC.ZIKV-ns4b-ns5/dN231 e IC.ZIKV-ns4b-ns5/N3, respectivamente. Estudos de infecção celular e em camundongos AG129 demonstraram que apenas IC.ZIKV-ns4b-ns5/N3 exibiu perfil menos proliferativo em modelos celulares e menos virulento em camundongos AG129, demonstrando um padrão atenuado em comparação ao vírus IC.ZIKV-Rio-U1. Além disso, o vírus IC.ZIKV-ns4b-ns5/N3 induziu imunidade em camundongos AG129, protegendo esses animais contra desafio letal com o vírus selvagem Rio-U1. Esses resultados demonstram que, a estratégia de desotimização de pares de códons pode ser uma ferramenta para o desenvolvimento de vírus atenuados e protótipos vacinais