Combination of surveillance tools reveals that Yellow Fever virus can remain in the same Atlantic Forest area at least for three transmission seasons.

BACKGROUND: In Brazil, the Yellow Fever virus (YFV) is endemic in the Amazon, from where it eventually expands into epidemic waves. Coastal south-eastern (SE) Brazil, which has been a YFV-free region for eight decades, has reported a severe sylvatic outbreak since 2016. The virus spread from the north toward the south of the Rio de Janeiro (RJ) state, causing 307 human cases with 105 deaths during the 2016-2017 and 2017-2018 transmission seasons. It is unclear, however, whether the YFV would persist in the coastal Atlantic Forest of RJ during subsequent transmission seasons. OBJECTIVES: To conduct a real-time surveillance and assess the potential persistence of YFV in the coastal Atlantic Forest of RJ during the 2018-2019 transmission season. METHODS: We combined epizootic surveillance with fast diagnostic and molecular, phylogenetic, and evolutionary analyses. FINDINGS: Using this integrative strategy, we detected the first evidence of YFV re-emergence in the third transmission season (2018-2019) in a dying howler monkey from the central region of the RJ state. The YFV detected in 2019 has the molecular signature associated with the current SE YFV outbreak and exhibited a close phylogenetic relationship with the YFV lineage that circulated in the same Atlantic Forest fragment during the past seasons. This lineage circulated along the coastal side of the Serra do Mar mountain chain, and its evolution seems to be mainly driven by genetic drift. The potential bridge vector Aedes albopictus was found probing on the recently dead howler monkey in the forest edge, very close to urban areas. MAIN CONCLUSIONS: Collectively, our data revealed that YFV transmission persisted at the same Atlantic Forest area for at least three consecutive transmission seasons without the need of new introductions. Our real-time surveillance strategy permitted health authorities to take preventive actions within 48 h after the detection of the sick non-human primate. The local virus persistence and the proximity of the epizootic forest to urban areas reinforces the concern with regards to the risk of re-urbanisation and seasonal re-emergence of YFV, stressing the need for continuous effective surveillance and high vaccination coverage in the SE region, particularly in RJ, an important tourist location.

Amino Acid Polymorphisms on the Brazilian Strain of Yellow Fever Virus Methyltransferase Are Related to the Host's Immune Evasion Mediated by Type I Interferon.

Since late 2016, a yellow fever virus (YFV) variant carrying a set of nine amino acid variations has circulated in South America. Three of them were mapped on the methyltransferase (MTase) domain of viral NS5 protein. To assess whether these changes affected viral infectivity, we synthesized YFV carrying the MTase of circulating lineage as well as its isoform with the residues of the previous strains (NS5 K101R, NS5 V138I, and NS5 G173S). We observed a slight difference in viral growth properties and plaque phenotype between the two synthetic YFVs. However, the MTase polymorphisms associated with the Brazilian strain of YFV (2016-2019) confer more susceptibility to the IFN-I. In addition, in vitro MTase assay revealed that the interaction between the YFV MTase and the methyl donor molecule (SAM) is altered in the Brazilian MTase variant. Altogether, the results reported here describe that the MTase carrying the molecular signature of the Brazilian YFV circulating since 2016 might display a slight decrease in its catalytic activity but virtually no effect on viral fitness in the parameters comprised in this study. The most marked influence of these residues stands in the immune escape against the antiviral response mediated by IFN-I.

Ecological, Genetic, and Phylogenetic Aspects of YFV 2017-2019 Spread in Rio de Janeiro State.

In Brazil, a yellow fever (YF) outbreak was reported in areas considered YF-free for decades. The low vaccination coverage and the increasing forest fragmentation, with the wide distribution of vector mosquitoes, have been related to yellow fever virus (YFV) transmission beyond endemic areas since 2016. Aiming to elucidate the molecular and phylogenetic aspects of YFV spread on a local scale, we generated 43 new YFV genomes sampled from humans, non-human primates (NHP), and primarily, mosquitoes from highly heterogenic areas in 15 localities from Rio de Janeiro (RJ) state during the YFV 2016-2019 outbreak in southeast Brazil. Our analysis revealed that the genetic diversity and spatial distribution of the sylvatic transmission of YFV in RJ originated from at least two introductions and followed two chains of dissemination, here named the YFV RJ-I and YFV RJ-II clades. They moved with similar dispersal speeds from the north to the south of the RJ state in parallel directions, separated by the Serra do Mar Mountain chain, with YFV RJ-I invading the north coast of São Paulo state. The YFV RJ-I clade showed a more significant heterogeneity across the entire polyprotein. The YFV RJ-II clade, with only two amino acid polymorphisms, mapped at NS1 (I1086V), present only in mosquitoes at the same locality and NS4A (I2176V), shared by all YFV clade RJ-II, suggests a recent clustering of YFV isolates collected from different hosts. Our analyses strengthen the role of surveillance, genomic analyses of YVF isolated from other hosts, and environmental studies into the strategies to forecast, control, and prevent yellow fever outbreaks.

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.

Genotype-specific features reduce the susceptibility of South American yellow fever virus strains to vaccine-induced antibodies.

The resurgence of yellow fever in South America has prompted vaccination against the etiologic agent, yellow fever virus (YFV). Current vaccines are based on a live-attenuated YF-17D virus derived from a virulent African isolate. The capacity of these vaccines to induce neutralizing antibodies against the vaccine strain is used as a surrogate for protection. However, the sensitivity of genetically distinct South American strains to vaccine-induced antibodies is unknown. We show that antiviral potency of the polyclonal antibody response in vaccinees is attenuated against an emergent Brazilian strain. This reduction was attributable to amino acid changes at two sites in central domain II of the glycoprotein E, including multiple changes at the domain I-domain II hinge, which are unique to and shared among most South American YFV strains. Our findings call for a reevaluation of current approaches to YFV immunological surveillance in South America and suggest approaches for updating vaccines.

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.

Structural characterization and polymorphism analysis of the NS2B-NS3 protease from the 2017 Brazilian circulating strain of Yellow Fever virus.

BACKGROUND: The Yellow Fever virus (YFV) is transmitted by mosquitos and causes an infection with symptoms including fever, headaches and nausea. In 20-50% of the cases, the disease may evolve to a visceral stage, reaching high mortality rates. YFV NS2B-NS3 protease has been identified as an important drug target. METHODS: Herein, we describe the crystal structure of the NS2B-NS3 protease from the 2017 YFV Brazilian circulating strain using X-ray crystallography. Furthermore, we used a combination of biochemical and biophysical assays to characterize the enzyme and investigate the impact of the polymorphisms observed in different YFV circulating strains. RESULTS: Surprisingly, the crystal structure of YFV protease seems to adopt the closed conformation without the presence of a binding partner. Although D88E and K121R mutants exhibited a lower affinity for the substrate, both revealed to be more processive, resulting in a similar catalytic efficiency in relation to the WT protease. Still, both mutants showed an accentuated decrease in stability when compared with the WT. CONCLUSIONS: The crystal structure of YFV NS2B-NS3 in closed conformation might be an important tool for the development of new drugs, as well as understanding the activation mechanism of viral proteases. Biochemical analyses indicate that the NS2B-NS3 protease of the circulating strain of YFV is more stable than previous strains. GENERAL SIGNIFICANCE: The YFV NS2B-NS3 protease is the first flaviviral structure described in its closed conformation when in a free form, implying that external factors might induce the activation of the enzyme.

Distinct YFV Lineages Co-circulated in the Central-Western and Southeastern Brazilian Regions From 2015 to 2018.

The current outbreak of yellow fever virus (YFV) that is afflicting Brazil since the end of 2016 probably originated from a re-introduction of YFV from endemic areas into the non-endemic Southeastern Brazil. However, the lack of genomic sequences from endemic regions hinders the tracking of YFV's dissemination routes. We assessed the origin and spread of the ongoing YFV Brazilian outbreak analyzing a new set of YFV strains infecting humans, non-human primates (NHPs) and mosquitoes sampled across five Brazilian states from endemic and non-endemic regions between 2015 and 2018. We found two YFV sub-clade 1E lineages circulating in NHP from Goiás state (GO), resulting from independent viral introductions into the Araguaia tributary river basin: while one strain from 2017 clustered intermingled with Venezuelan YFV strains from 2000, the other YFV strains sampled in 2015 and 2017 clustered with sequences of the current YFV outbreak in the Brazilian Southeastern region (named YFV2015-2018 lineage), displaying the same molecular signature associated to the current YFV outbreak. After its introduction in GO at around mid-2014, the YFV2015-2018 lineage followed two paths of dissemination outside GO, originating two major YFV sub-lineages: (1) the YFVMG/ES/RJ sub-lineage spread sequentially from the eastern area of Minas Gerais state to Espírito Santo and then to Rio de Janeiro states, following the Southeast Atlantic basin; (2) the YFVMG/SP sub-lineage spread from the southwestern area of Minas Gerais to the metropolitan region of São Paulo state, following the Paraná basin. These results indicate the ongoing YFV outbreak in Southeastern Brazil originated from a dissemination event from GO almost 2 years before its recognition at the end of 2016. From GO this lineage was introduced in Minas Gerais state at least two times, originating two sub-lineages that followed different routes toward densely populated areas. The spread of YFV outside endemic regions for at least 4 years stresses the imperative importance of the continuous monitoring of YFV to aid decision-making for effective control policies aiming the increase of vaccination coverage to avoid the YFV transmission in densely populated urban centers.

Combination of surveillance tools reveals that Yellow Fever virus can remain in the same Atlantic Forest area at least for three transmission seasons

BACKGROUND In Brazil, the Yellow Fever virus (YFV) is endemic in the Amazon, from where it eventually expands into epidemic waves. Coastal south-eastern (SE) Brazil, which has been a YFV-free region for eight decades, has reported a severe sylvatic outbreak since 2016. The virus spread from the north toward the south of the Rio de Janeiro (RJ) state, causing 307 human cases with 105 deaths during the 2016-2017 and 2017-2018 transmission seasons. It is unclear, however, whether the YFV would persist in the coastal Atlantic Forest of RJ during subsequent transmission seasons. OBJECTIVES To conduct a real-time surveillance and assess the potential persistence of YFV in the coastal Atlantic Forest of RJ during the 2018-2019 transmission season. METHODS We combined epizootic surveillance with fast diagnostic and molecular, phylogenetic, and evolutionary analyses. FINDINGS Using this integrative strategy, we detected the first evidence of YFV re-emergence in the third transmission season (2018-2019) in a dying howler monkey from the central region of the RJ state. The YFV detected in 2019 has the molecular signature associated with the current SE YFV outbreak and exhibited a close phylogenetic relationship with the YFV lineage that circulated in the same Atlantic Forest fragment during the past seasons. This lineage circulated along the coastal side of the Serra do Mar mountain chain, and its evolution seems to be mainly driven by genetic drift. The potential bridge vector Aedes albopictus was found probing on the recently dead howler monkey in the forest edge, very close to urban areas. MAIN CONCLUSIONS Collectively, our data revealed that YFV transmission persisted at the same Atlantic Forest area for at least three consecutive transmission seasons without the need of new introductions. Our real-time surveillance strategy permitted health authorities to take preventive actions within 48 h after the detection of the sick non-human primate. The local virus persistence and the proximity of the epizootic forest to urban areas reinforces the concern with regards to the risk of re-urbanisation and seasonal re-emergence of YFV, stressing the need for continuous effective surveillance and high vaccination coverage in the SE region, particularly in RJ, an important tourist location.

Estudo de mutações nas proteínas virais E, NS3 e NS4B no genoma do vírus vacinal da Febre Amarela 17D / Study of mutations in the viral proteins E, NS3 and NS4B in the genome of the yellow fever vaccine virus 17D

Resumo: A Febre Amarela (FA) é uma doença infecciosa não contagiosa, causada por um arbovírus e objeto de preocupação sanitária mundial. A principal medida de controle é a vacinação com o vírus atenuado da FA, cepa 17D, que é capaz de induzir resposta imune protetora a longo prazo com administração em dose única. Vírus atenuados são potentes vetores de expressão, pois disseminam o antígeno no hospedeiro e induzem resposta imune protetora, contribuindo para o desenvolvimento de vacinas recombinantes. Diversas estratégias foram desenvolvidas para expressão de genes heterólogos pelo vírus vacinal FA 17D. Entretanto, a inserção induz proliferação viral e imunogenicidade reduzidas. Neste trabalho foi utilizado o vírus vacinal FA 17D recombinante expressando a proteína repórter enhanced green fluorescent protein (EGFP) para avaliar o impacto de mutações específicas que possam modular a replicação viral. Mutações nas proteínas E, NS3 e NS4B foram descritas por aumentarem a proliferação viral em cultura de células e em camundongos, no genoma do vírus da dengue, sorotipos 1 e 2. As mutações em E400 (F→L), E403 (T→I), NS3439 (V→S) e NS4B54 (L→F) foram inseridas no genoma do vírus FA/EGFP, com a finalidade de caracterizar o seu efeito na proliferação viral e na indução de resposta imune humoral. O cDNA do genoma viral FA/EGFP foi utilizado para gerar vírus recombinantes carreando uma, duas ou três mutações. O estudo de proliferação viral foi realizado por cinética de infecção de células das linhagens Vero, Huh7 e C6/36. Os resultados mostram que os vírus da FA recombinantes se proliferam menos que o vírus vacinal FA 17DD. Além disso, a infectividade dos vírus mutantes em células de mamífero é diferente da infectividade em células de mosquitoOs vírus que carreiam as mutações em E400/NS3439 e E400/NS3439/NS4B54 tem a proliferação viral significativamente prejudicada em células de mamífero. Os vírus que carreiam a mutação em E400 apresentaram aumento de proliferação viral em comparação com o vírus FA/EGFP original, em células de mosquito. As diferenças entre os tipos celulares podem ter sido causadas pelas características fisiológicas das células durante a infecção viral e pelas diferenças de propriedades das proteínas virais ocasionadas pela inserção das mutações. Não foi possível recuperar partículas virais infecciosas carreando a mutação em E403. A modelagem molecular das proteínas virais mostrou diferenças discretas de carga, volume de superfície proteica e propriedade físico-química induzidas pelas mutações. Nenhuma das mutações influenciou nas interações intramoleculares. A imunogenicidade foi avaliada por imunização de camundongos das linhagens C57BL/6 e BALB/c com os vírus carreando mutações únicas e os soros foram analisados por PRNT e ELISA para obter os títulos de anticorpos neutralizantes para FA e anticorpos para GFP, respectivamente. Os soros dos camundongos imunizados com os vírus recombinantes apresentam menores títulos de anticorpos neutralizantes em comparação ao grupo imunizado com o vírus vacinal, porém não houveram alterações na indução de anticorpos para GFP. De maneira geral, as mutações em E400 e E403 produziram maiores efeitos sobre a proliferação viral e as mutações NS3439 e NS4B54, na imunogenicidade. (AU)