RESUMO
São Paulo, a densely inhabited state in southeast Brazil that contains the fourth most populated city in the world, recently experienced its largest yellow fever virus (YFV) outbreak in decades. YFV does not normally circulate extensively in São Paulo, so most people were unvaccinated when the outbreak began. Surveillance in non-human primates (NHPs) is important for determining the magnitude and geographic extent of an epizootic, thereby helping to evaluate the risk of YFV spillover to humans. Data from infected NHPs can give more accurate insights into YFV spread than when using data from human cases alone. To contextualise human cases, identify epizootic foci and uncover the rate and direction of YFV spread in São Paulo, we generated and analysed virus genomic data and epizootic case data from NHPs in São Paulo. We report the occurrence of three spatiotemporally distinct phases of the outbreak in São Paulo prior to February 2018. We generated 51 new virus genomes from YFV positive cases identified in 23 different municipalities in São Paulo, mostly sampled from NHPs between October 2016 and January 2018. Although we observe substantial heterogeneity in lineage dispersal velocities between phylogenetic branches, continuous phylogeographic analyses of generated YFV genomes suggest that YFV lineages spread in São Paulo at a mean rate of approximately 1km per day during all phases of the outbreak. Viral lineages from the first epizootic phase in northern São Paulo subsequently dispersed towards the south of the state to cause the second and third epizootic phases there. This alters our understanding of how YFV was introduced into the densely populated south of São Paulo state. Our results shed light on the sylvatic transmission of YFV in highly fragmented forested regions in São Paulo state and highlight the importance of continued surveillance of zoonotic pathogens in sentinel species.
Assuntos
Genoma Viral , Doenças dos Primatas/virologia , Febre Amarela/veterinária , Febre Amarela/virologia , Vírus da Febre Amarela/genética , Zoonoses/virologia , Animais , Brasil/epidemiologia , Surtos de Doenças , Genômica , Humanos , Filogenia , Filogeografia , Doenças dos Primatas/epidemiologia , Doenças dos Primatas/transmissão , Primatas/virologia , Febre Amarela/epidemiologia , Febre Amarela/transmissão , Vírus da Febre Amarela/classificação , Vírus da Febre Amarela/isolamento & purificação , Zoonoses/epidemiologia , Zoonoses/transmissãoRESUMO
Yellow fever virus (YFV) causes a clinical syndrome of acute hemorrhagic hepatitis. YFV transmission involves non-human primates (NHP), mosquitoes and humans. By late 2016, Brazil experienced the largest YFV outbreak of the last 100 years, with 2050 human confirmed cases, with 681 cases ending in death and 764 confirmed epizootic cases in NHP. Among affected areas, Bahia state in Northeastern was the only region with no autochthonous human cases. By using next generation sequence approach, we investigated the molecular epidemiology of YFV in NHP in Bahia and discuss what factors might have prevented human cases. We investigated 47 YFV positive tissue samples from NHP cases to generate 8 novel YFV genomes. ML phylogenetic tree reconstructions and automated subtyping tools placed the newly generated genomes within the South American genotype I (SA I). Our analysis revealed that the YFV genomes from Bahia formed two distinct well-supported phylogenetic clusters that emerged most likely of an introduction from Minas Gerais and Espírito Santo states. Vegetation coverage analysis performed shows predominantly low to medium vegetation coverage in Bahia state. Together, our findings support the hypothesis of two independent YFV SA-I introductions. We also highlighted the effectiveness of the actions taken by epidemiological surveillance team of the state to prevented human cases.
Assuntos
Doenças dos Primatas/virologia , Febre Amarela/veterinária , Vírus da Febre Amarela/genética , Alouatta , Animais , Brasil/epidemiologia , Callithrix , Ecossistema , Genoma Viral , Humanos , Filogenia , Febre Amarela/epidemiologia , Febre Amarela/prevenção & controle , Febre Amarela/transmissão , Vírus da Febre Amarela/classificaçãoRESUMO
One patient presented hyporexia, asthenia, adynamia, and jaundice two months after acute yellow fever (YF) onset; plus laboratory tests indicating hepatic cytolysis and a rebound of alanine and aspartate transaminases, and total and direct bilirubin levels. Laboratory tests discarded autoimmune hepatitis, inflammatory or metabolic liver disease, and new infections caused by hepatotropic agents. Anti-YFV IgM, IgG and neutralizing antibodies were detected in different times, but no viremia. A liver biopsy was collected three months after YF onset and tested positive for YFV antigens and wild-type YFV-RNA (364 RNA-copies/gram/liver). Transaminases and bilirubin levels remained elevated for five months, and the arresting of symptoms persisted for six months after the acute YF onset. Several serum chemokines, cytokines, and growth factors were measured. A similar immune response profile was observed in the earlier phases of the disease, followed by more pronounced changes in the later stages, when transaminases levels returned to normal. The results indicated viral persistence in the liver and continual liver cell damage three months after YF onset and reinforced the need for extended follow-ups of YF patients. Further studies to investigate the role of possible viral persistence and the immune response causing relapsing hepatitis following YF are also necessary.
Assuntos
Anticorpos Antivirais/sangue , Hepatite A/diagnóstico , Fígado/virologia , Febre Amarela/complicações , Doença Aguda , Anticorpos Neutralizantes/sangue , Biópsia , Citocinas/sangue , Hepatite A/imunologia , Humanos , Icterícia/virologia , Fígado/patologia , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Recidiva , Fatores de Tempo , Vírus da Febre Amarela/classificação , Vírus da Febre Amarela/imunologiaRESUMO
The recent reemergence of yellow fever virus (YFV) in Brazil has raised serious concerns due to the rapid dissemination of the virus in the southeastern region. To better understand YFV genetic diversity and dynamics during the recent outbreak in southeastern Brazil, we generated 18 complete and nearly complete genomes from the peak of the epidemic curve from nonhuman primates (NHPs) and human infected cases across the Espírito Santo and Rio de Janeiro states. Genomic sequencing of 18 YFV genomes revealed the estimated timing, source, and likely routes of yellow fever virus transmission and dispersion during one of the largest outbreaks ever registered in Brazil. We showed that during the recent epidemic, YFV was reintroduced from Minas Gerais to the Espírito Santo and Rio de Janeiro states multiple times between 2016 and 2019. The analysis of data from portable sequencing could identify the corridor of spread of YFV. These findings reinforce the idea that continued genomic surveillance strategies can provide information on virus genetic diversity and transmission dynamics that might assist in understanding arbovirus epidemics.IMPORTANCE Arbovirus infections in Brazil, including yellow fever, dengue, zika, and chikungunya, result in considerable morbidity and mortality and are pressing public health concerns. However, our understanding of these outbreaks is hampered by the limited availability of genomic data. In this study, we investigated the genetic diversity and spatial distribution of YFV during the current outbreak by analyzing genomic data from areas in southeastern Brazil not covered by other previous studies. To gain insights into the routes of YFV introduction and dispersion, we tracked the virus by sequencing YFV genomes sampled from nonhuman primates and infected patients from the southeastern region. Our study provides an understanding of how YFV initiates transmission in new Brazilian regions and illustrates that genomics in the field can augment traditional approaches to infectious disease surveillance and control.
Assuntos
Surtos de Doenças , Genoma Viral , Febre Amarela/epidemiologia , Febre Amarela/transmissão , Vírus da Febre Amarela/genética , Aedes/virologia , Alouatta/virologia , Animais , Brasil/epidemiologia , Callithrix/virologia , Cebus/virologia , Feminino , Variação Genética , Humanos , Incidência , Leontopithecus/virologia , Masculino , Mosquitos Vetores/virologia , Filogenia , Filogeografia , Sequenciamento Completo do Genoma , Febre Amarela/virologia , Vírus da Febre Amarela/classificação , Vírus da Febre Amarela/isolamento & purificação , Vírus da Febre Amarela/patogenicidadeRESUMO
OBJECTIVES: Yellow fever virus historically was a frequent threat to American and European coasts. Medical milestones such as the discovery of mosquitoes as vectors and subsequently an effective vaccine significantly reduced its incidence, in spite of which, thousands of cases of this deathly disease still occur regularly in Sub-Saharan Africa and the Amazonian basin in South America, which are usually not reported. An urban outbreak in Angola, consecutive years of increasing incidence near major Brazilian cities, and imported cases in China, South America and Europe, have brought this virus back to the global spotlight. The aim of this article is to underline that the preventive YFV measures, such as vaccination, need to be carefully revised in order to minimize the risks of new YFV outbreaks, especially in urban or immunologically vulnerable places. Furthermore, this article highlights the diverse factors that have favored the spread of other Aedes spp.-associated arboviral diseases like Dengue, Chikungunya and Zika, to northern latitudes causing epidemics in the United States and Europe, emphasizing the possibility that YFV might follow the path of these viruses unless enhanced surveillance and efficient control systems are urgently initiated.
Assuntos
Febre Amarela/epidemiologia , Vírus da Febre Amarela/isolamento & purificação , Animais , Humanos , Mosquitos Vetores/fisiologia , Mosquitos Vetores/virologia , América do Norte/epidemiologia , Febre Amarela/transmissão , Febre Amarela/virologia , Vírus da Febre Amarela/classificação , Vírus da Febre Amarela/genéticaRESUMO
We report a 3-year-old child who was hospitalized because of severe manifestations of the central nervous system. The child died after 6 days of hospitalization. Analysis of postmortem cerebrospinal fluid showed the presence of yellow fever virus RNA. Nucleotide sequencing confirmed that the virus was wild-type yellow fever virus.
Assuntos
RNA Viral/genética , Febre Amarela/líquido cefalorraquidiano , Febre Amarela/virologia , Vírus da Febre Amarela/genética , Antivirais/farmacologia , Antivirais/uso terapêutico , Autopsia , Biomarcadores , Brasil , Pré-Escolar , Progressão da Doença , Evolução Fatal , Feminino , Humanos , Filogenia , Análise de Sequência de DNA , Avaliação de Sintomas , Tomografia Computadorizada por Raios X , Febre Amarela/diagnóstico , Febre Amarela/tratamento farmacológico , Vírus da Febre Amarela/classificação , Vírus da Febre Amarela/isolamento & purificaçãoRESUMO
Beginning in late 2016 Brazil faced the worst outbreak of Yellow Fever in recent decades, mainly located in southeastern rural regions of the country. In the present study we characterize the Yellow Fever Virus (YFV) associated with this outbreak in São Paulo State, Brazil. Blood or tissues collected from 430 dead monkeys and 1030 pools containing a total of 5,518 mosquitoes were tested for YFV by quantitative RT-PCR, immunohistochemistry (IHC) and indirect immunofluorescence. A total of 67 monkeys were YFV-positive and 3 pools yielded YFV following culture in a C6/36 cell line. Analysis of five nearly full length genomes of YFV from collected samples was consistent with evidence that the virus associated with the São Paulo outbreak originated in Minas Gerais. The phylogenetic analysis also showed that strains involved in the 2016-2017 outbreak in distinct Brazilian states (i.e., Minas Gerais, Rio de Janeiro, Espirito Santo) intermingled in maximum-likelihood and Bayesian trees. Conversely, the strains detected in São Paulo formed a monophyletic cluster, suggesting that they were local-adapted. The finding of YFV by RT-PCR in five Callithrix monkeys who were all YFV-negative by histopathology or immunohistochemistry suggests that this YFV lineage circulating in Sao Paulo is associated with different outcomes in Callithrix when compared to other monkeys.
Assuntos
Culicidae/virologia , Surtos de Doenças/classificação , Haplorrinos/virologia , Febre Amarela/epidemiologia , Vírus da Febre Amarela/classificação , Animais , Antígenos Virais/análise , Teorema de Bayes , Brasil/epidemiologia , Linhagem Celular , Humanos , Filogenia , Filogeografia , Sequenciamento Completo do Genoma , Febre Amarela/imunologia , Febre Amarela/virologia , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/imunologia , Vírus da Febre Amarela/isolamento & purificação , Zoonoses/virologiaRESUMO
The yellow fever virus (YFV) epidemic in Brazil is the largest in decades. The recent discovery of YFV in Brazilian Aedes species mosquitos highlights a need to monitor the risk of reestablishment of urban YFV transmission in the Americas. We use a suite of epidemiological, spatial, and genomic approaches to characterize YFV transmission. We show that the age and sex distribution of human cases is characteristic of sylvatic transmission. Analysis of YFV cases combined with genomes generated locally reveals an early phase of sylvatic YFV transmission and spatial expansion toward previously YFV-free areas, followed by a rise in viral spillover to humans in late 2016. Our results establish a framework for monitoring YFV transmission in real time that will contribute to a global strategy to eliminate future YFV epidemics.
Assuntos
Surtos de Doenças/prevenção & controle , Monitoramento Epidemiológico , Genômica/métodos , Febre Amarela/prevenção & controle , Febre Amarela/transmissão , Vírus da Febre Amarela/isolamento & purificação , Aedes/virologia , Fatores Etários , Animais , Brasil/epidemiologia , Surtos de Doenças/estatística & dados numéricos , Evolução Molecular , Humanos , Filogenia , Reação em Cadeia da Polimerase , Risco , Fatores Sexuais , Análise Espaço-Temporal , Febre Amarela/epidemiologia , Febre Amarela/virologia , Vírus da Febre Amarela/classificação , Vírus da Febre Amarela/genéticaRESUMO
Southeastern Brazil has been suffering a rapid expansion of a severe sylvatic yellow fever virus (YFV) outbreak since late 2016, which has reached one of the most populated zones in Brazil and South America, heretofore a yellow fever-free zone for more than 70 years. In the current study, we describe the complete genome of 12 YFV samples from mosquitoes, humans and non-human primates from the Brazilian 2017 epidemic. All of the YFV sequences belong to the modern lineage (sub-lineage 1E) of South American genotype I, having been circulating for several months prior to the December 2016 detection. Our data confirm that viral strains associated with the most severe YF epidemic in South America in the last 70 years display unique amino acid substitutions that are mainly located in highly conserved positions in non-structural proteins. Our data also corroborate that YFV has spread southward into Rio de Janeiro state following two main sylvatic dispersion routes that converged at the border of the great metropolitan area comprising nearly 12 million unvaccinated inhabitants. Our original results can help public health authorities to guide the surveillance, prophylaxis and control measures required to face such a severe epidemiological problem. Finally, it will also inspire other workers to further investigate the epidemiological and biological significance of the amino acid polymorphisms detected in the Brazilian 2017 YFV strains.
Assuntos
Febre Amarela/virologia , Vírus da Febre Amarela/genética , Brasil/epidemiologia , Surtos de Doenças , Genoma Viral , Genômica , Genótipo , Humanos , Modelos Moleculares , Filogenia , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Febre Amarela/epidemiologia , Vírus da Febre Amarela/química , Vírus da Febre Amarela/classificação , Vírus da Febre Amarela/isolamento & purificaçãoRESUMO
In January 2017, a yellow fever outbreak occurred in Espirito Santo, Brazil, where human immunization coverage is low. Histologic, immunohistologic, and PCR examinations were performed for 22 deceased nonhuman New World primates; typical yellow fever features were found in 21. Diagnosis in nonhuman primates prompted early public health response.
Assuntos
Surtos de Doenças , Doenças dos Primatas/epidemiologia , RNA Viral/genética , Febre Amarela/epidemiologia , Febre Amarela/veterinária , Vírus da Febre Amarela/genética , Animais , Brasil/epidemiologia , Haplorrinos/virologia , Coração/fisiopatologia , Coração/virologia , Humanos , Rim/patologia , Rim/virologia , Fígado/patologia , Fígado/virologia , Pulmão/patologia , Pulmão/virologia , Doenças dos Primatas/transmissão , Doenças dos Primatas/virologia , Baço/patologia , Baço/virologia , Febre Amarela/transmissão , Febre Amarela/virologia , Vírus da Febre Amarela/classificação , Vírus da Febre Amarela/isolamento & purificação , Vírus da Febre Amarela/patogenicidadeRESUMO
Yellow fever virus (YFV) strains circulating in the Americas belong to two distinct genotypes (I and II) that have diversified into several concurrent enzootic lineages. Since 1999, YFV genotype I has spread outside endemic regions and its recent (2017) reemergence in non-endemic Southeastern Brazilian states fuels one of the largest epizootic of jungle Yellow Fever registered in the country. To better understand this phenomenon, we reconstructed the phylodynamics of YFV American genotypes using sequences from nine countries sampled along 60 years, including strains from Brazilian 2017 outbreak. Our analyses reveals that YFV genotypes I and II follow roughly similar evolutionary and demographic dynamics until the early 1990s, when a dramatic change in the diversification process of the genotype I occurred associated with the emergence and dissemination of a new lineage (here called modern). Trinidad and Tobago was the most likely source of the YFV modern-lineage that spread to Brazil and Venezuela around the late 1980s, where it replaced all lineages previously circulating. The modern-lineage caused all major YFV outbreaks detected in non-endemic South American regions since 2000, including the 2017 Brazilian outbreak, and its dissemination was coupled to the accumulation of several amino acid substitutions particularly within non-structural viral proteins.
Assuntos
Surtos de Doenças , Febre Amarela/epidemiologia , Vírus da Febre Amarela/genética , Brasil/epidemiologia , Evolução Molecular , Genótipo , Humanos , Filogenia , Filogeografia , Trinidad e Tobago , Proteínas Virais/genética , Febre Amarela/virologia , Vírus da Febre Amarela/classificaçãoRESUMO
This randomised, double-blind, multicentre study with children nine-23 months old evaluated the immunogenicity of yellow fever (YF) vaccines prepared with substrains 17DD and 17D-213/77. YF antibodies were titered before and 30 or more days after vaccination. Seropositivity and seroconversion were analysed according to the maternal serological status and the collaborating centre. A total of 1,966 children were randomised in the municipalities of the states of Mato Grosso do Sul, Minas Gerais and São Paulo and blood samples were collected from 1,714 mothers. Seropositivity was observed in 78.6% of mothers and 8.9% of children before vaccination. After vaccination, seropositivity rates of 81.9% and 83.2%, seroconversion rates of 84.8% and 85.8% and rates of a four-fold increase over the pre-vaccination titre of 77.6% and 81.8% were observed in the 17D-213/77 and 17DD subgroups, respectively. There was no association with maternal immunity. Among children aged 12 months or older, the seroconversion rates of 69% were associated with concomitant vaccination against measles, mumps and rubella. The data were not conclusive regarding the interference of maternal immunity in the immune response to the YF vaccine, but they suggest interference from other vaccines. The failures in seroconversion after vaccination support the recommendation of a booster dose in children within 10 years of the first dose.
Assuntos
Anticorpos Antivirais/isolamento & purificação , Antivirais/uso terapêutico , Soroconversão , Vacina contra Febre Amarela/imunologia , Febre Amarela/prevenção & controle , Vírus da Febre Amarela/imunologia , Anticorpos Neutralizantes , Causalidade , Diarreia/etiologia , Método Duplo-Cego , Feminino , Febre/etiologia , Técnica de Placa Hemolítica , Rouquidão/etiologia , Humanos , Lactente , Masculino , Convulsões/etiologia , Resultado do Tratamento , Vômito/etiologia , Vacina contra Febre Amarela/efeitos adversos , Vírus da Febre Amarela/classificaçãoRESUMO
This randomised, double-blind, multicentre study with children nine-23 months old evaluated the immunogenicity of yellow fever (YF) vaccines prepared with substrains 17DD and 17D-213/77. YF antibodies were tittered before and 30 or more days after vaccination. Seropositivity and seroconversion were analysed according to the maternal serological status and the collaborating centre. A total of 1,966 children were randomised in the municipalities of the states of Mato Grosso do Sul, Minas Gerais and São Paulo and blood samples were collected from 1,714 mothers. Seropositivity was observed in 78.6% of mothers and 8.9% of children before vaccination. After vaccination, seropositivity rates of 81.9% and 83.2%, seroconversion rates of 84.8% and 85.8% and rates of a four-fold increase over the pre-vaccination titre of 77.6% and 81.8% were observed in the 17D-213/77 and 17DD subgroups, respectively. There was no association with maternal immunity. Among children aged 12 months or older, the seroconversion rates of 69% were associated with concomitant vaccination against measles, mumps and rubella. The data were not conclusive regarding the interference of maternal immunity in the immune response to the YF vaccine, but they suggest interference from other vaccines. The failures in seroconversion after vaccination support the recommendation of a booster dose in children within 10 years of the first dose.
Assuntos
Humanos , Masculino , Feminino , Lactente , Anticorpos Antivirais/isolamento & purificação , Antivirais/uso terapêutico , Soroconversão , Vacina contra Febre Amarela/imunologia , Vírus da Febre Amarela/imunologia , Febre Amarela/prevenção & controle , Anticorpos Neutralizantes , Causalidade , Diarreia/etnologia , Método Duplo-Cego , Febre/etnologia , Técnica de Placa Hemolítica , Rouquidão/etnologia , Convulsões/etnologia , Resultado do Tratamento , Vômito/etnologia , Vacina contra Febre Amarela/efeitos adversos , Vírus da Febre Amarela/classificaçãoRESUMO
Yellow fever virus (YFV) is historically one of the most important viruses to affect human populations. Despite the existence of highly effective vaccines for over 70 years, yellow fever remains a significant and re-emerging cause of morbidity and mortality in endemic and high-risk regions of South America and Africa. The virus may be maintained in sylvatic enzootic/epizootic, transitional and urban epidemic transmission cycles with geographic variation in terms of levels of genetic diversity, the nature of transmission cycles and patterns of outbreak activity. In this review we consider evolutionary and ecological factors underlying YFV emergence, maintenance and spread, geographic distribution and patterns of epizootic/epidemic activity.
Assuntos
Evolução Molecular , Febre Amarela/epidemiologia , Vírus da Febre Amarela/genética , Aedes/virologia , África , Animais , Surtos de Doenças , Genes Virais , Genótipo , Humanos , Filogenia , Filogeografia , América do Sul , Febre Amarela/transmissão , Vírus da Febre Amarela/classificaçãoRESUMO
Globally, yellow fever virus infects nearly 200,000 people, leading to 30,000 deaths annually. Although the virus is endemic to Latin America, only a single genome from this region has been sequenced. Here, we report 12 Brazilian yellow fever virus complete genomes, their genetic traits, phylogenetic characterization, and phylogeographic dynamics. Variable 3' noncoding region (3'NCR) patterns and specific mutations throughout the open reading frame altered predicted secondary structures. Our findings suggest that whereas the introduction of yellow fever virus in Brazil led to genotype I-predominant dispersal throughout South and Central Americas, genotype II remained confined to Bolivia, Peru, and the western Brazilian Amazon.
Assuntos
Genoma Viral , Filogenia , Vírus da Febre Amarela/genética , Sequência de Bases , Brasil , Primers do DNA , Glicosilação , Reação em Cadeia da Polimerase , Vírus da Febre Amarela/classificaçãoRESUMO
Yellow fever virus (YFV) was isolated from Haemagogus leucocelaenus mosquitoes during an epizootic in 2001 in the Rio Grande do Sul State in southern Brazil. In October 2008, a yellow fever outbreak was reported there, with nonhuman primate deaths and human cases. This latter outbreak led to intensification of surveillance measures for early detection of YFV and support for vaccination programs. We report entomologic surveillance in 2 municipalities that recorded nonhuman primate deaths. Mosquitoes were collected at ground level, identified, and processed for virus isolation and molecular analyses. Eight YFV strains were isolated (7 from pools of Hg. leucocelaenus mosquitoes and another from Aedes serratus mosquitoes); 6 were sequenced, and they grouped in the YFV South American genotype I. The results confirmed the role of Hg. leucocelaenus mosquitoes as the main YFV vector in southern Brazil and suggest that Ae. serratus mosquitoes may have a potential role as a secondary vector.
Assuntos
Culicidae/virologia , Monitoramento Ambiental , Insetos Vetores/virologia , Febre Amarela/epidemiologia , Vírus da Febre Amarela/isolamento & purificação , Aedes/virologia , Animais , Animais Recém-Nascidos , Brasil/epidemiologia , Chlorocebus aethiops , Culicidae/classificação , Monitoramento Epidemiológico , Genes Virais/genética , Humanos , Insetos Vetores/classificação , Camundongos , Filogenia , Densidade Demográfica , População Rural , Células Vero , Febre Amarela/prevenção & controle , Febre Amarela/transmissão , Vírus da Febre Amarela/classificação , Vírus da Febre Amarela/genéticaRESUMO
Nucleotide sequences of two regions of the genomes of 11 yellow fever virus (YFV) samples isolated from monkeys or humans with symptomatic yellow fever (YF) in Brazil in 2000, 2004, and 2008 were determined with the objective of establishing the genotypes and studying the genetic variation. Results of the Bayesian phylogenetic analysis showed that sequences generated from strains from 2004 and 2008 formed a new subclade within the clade 1 of the South American genotype I. The new subgroup is here designated as 1E. Sequences of YFV strains recovered in 2000 belong to the subclade 1D, which comprises previously characterized YFV strains from Brazil. Molecular dating analyses suggested that the new subclade 1E started diversifying from 1D about 1975 and that the most recent 2004-2008 isolates arose about 1985.
Assuntos
Variação Genética , Doenças dos Macacos/epidemiologia , Filogenia , Febre Amarela/epidemiologia , Vírus da Febre Amarela , Regiões 3' não Traduzidas/genética , Animais , Teorema de Bayes , Brasil/epidemiologia , Evolução Molecular , Genótipo , Humanos , Dados de Sequência Molecular , Doenças dos Macacos/virologia , Análise de Sequência de DNA , América do Sul , Proteínas do Envelope Viral , Febre Amarela/veterinária , Febre Amarela/virologia , Vírus da Febre Amarela/classificação , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/isolamento & purificaçãoRESUMO
Um dos parâmetros principais para o estudo de atenuação de flavivírus é a determinação de sua capacidade de replicação. Neste contexto, empregamos o método de RT-PCR em tempo real que permite a detecção de ácidos nucléicos diretamente de amostras biológicas. Neste trabalho, utilizamos, então, esta tecnologia, para determinar a replicação de vírus de febre amarela 17DD e 17D-recombinantes, que expressam a proteína do envelope dos vírus dengue para validação destes como candidatos a vacinas. Esta metodologia foi empregada de forma complementar ao teste clássico de titulação viral por plaqueamento em culturas de células de vertebrados. Os materiais clínicos incluem soros de macacos rhesus previamente inoculados com estes vírus por via intracerebral ou subcutânea e soros de 32 indivíduos vacinados com vírus 17DD. O método demonstrou boa reprodutibilidade para amostras com título viral de até 2 Log10 PFU/mL com limite de detecção de até 10 PFU/mL ou 143 cópias /mL, sendo esta sensibilidade comparável ao que foi descrito para outros vírus analisados pela mesma tecnologia. Nesta faixa de concentração, os resultados obtidos pela metodologia de RT-PCR em tempo real mostraram-se compatíveis com os obtidos pelas técnicas de RT-PCR semi nested e pelo plaqueamento em soros de macacos inoculados com vírus 17DD. A análise comparativa da quantificação viral em espécimes clínicos de macacos sugere a limitada capacidade de replicação do vírus 17DD independente da via de inoculação. Os vírus 17D-recombinantes, avaliados neste estudo, demonstraram sua atenuação em relação ao vírus 17DD, tanto pela metodologia de RT-PCR em tempo real como por plaqueamento. Assim sendo, consideramos que a tecnologia empregada para o estudo da capacidade replicativa dos vírus 17DD e 17D-recombinantes em diferentes hospedeiros, demonstrou o perfil de atenuação dos vírus testados. Com relação aos vírus recombinantes 17D-dengue, estes resultados embasam a continuação do seu desenvolvimento como cand...
Assuntos
Humanos , Animais , Febre Amarela/epidemiologia , Reação em Cadeia da Polimerase , Vacina contra Febre Amarela/imunologia , Viremia/virologia , Vírus da Febre Amarela/classificaçãoRESUMO
The 3' noncoding region (3' NCR) of flaviviruses contains secondary and tertiary structures essential for virus replication. Previous studies of yellow fever virus (YFV) and dengue virus have found that modifications to the 3' NCR are sometimes associated with attenuation in vertebrate and/or mosquito hosts. The 3' NCRs of 117 isolates of South American YFV have been examined, and major deletions and/or duplications of conserved RNA structures have been identified in several wild-type isolates. Nineteen isolates (designated YF-XL isolates) from Brazil, Trinidad, and Venezuela, dating from 1973 to 2001, exhibited a 216-nucleotide (nt) duplication, yielding a tandem repeat of conserved hairpin, stem-loop, dumbbell, and pseudoknot structures. YF-XL isolates were found exclusively within one subclade of South American genotype I YFV. One Brazilian isolate exhibited, in addition to the 216-nt duplication, a deletion of a 40-nt repeated hairpin (RYF) motif (YF-XL-DeltaRYF). To investigate the biological significance of these 3' NCR rearrangements, YF-XL-DeltaRYF and YF-XL isolates, as well as other South American YFV isolates, were evaluated for three phenotypes: growth kinetics in cell culture, neuroinvasiveness in suckling mice, and ability to replicate and produce disseminated infections in Aedes aegypti mosquitoes. YF-XL-DeltaRYF and YF-XL isolates showed growth kinetics and neuroinvasive characteristics comparable to those of typical South American YFV isolates, and mosquito infectivity trials demonstrated that both types of 3' NCR variants were capable of replication and dissemination in a laboratory-adapted colony of A. aegypti.
Assuntos
Camundongos , Animais , Humanos , Research Support, Non-U.S. Gov't , Research Support, U.S. Gov't, P.H.S. , Aedes/virologia , Sequência de Bases , Células Cultivadas , Variação Genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , RNA não Traduzido/química , RNA não Traduzido/genética , RNA não Traduzido/fisiologia , RNA Viral/química , RNA Viral/genética , RNA Viral/fisiologia , Vírus da Febre Amarela/classificação , Vírus da Febre Amarela/crescimento & desenvolvimento , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/isolamento & purificação , Vírus da Febre Amarela/patogenicidade , Trinidad e Tobago , Brasil , VenezuelaRESUMO
The 3' noncoding region (3' NCR) of flaviviruses contains secondary and tertiary structures essential for virus replication. Previous studies of yellow fever virus (YFV) and dengue virus have found that modifications to the 3' NCR are sometimes associated with attenuation in vertebrate and/or mosquito hosts. The 3' NCRs of 117 isolates of South American YFV have been examined, and major deletions and/or duplications of conserved RNA structures have been identified in several wild-type isolates. Nineteen isolates (designated YF-XL isolates) from Brazil, Trinidad, and Venezuela, dating from 1973 to 2001, exhibited a 216-nucleotide (nt) duplication, yielding a tandem repeat of conserved hairpin, stem-loop, dumbbell, and pseudoknot structures. YF-XL isolates were found exclusively within one subclade of South American genotype I YFV. One Brazilian isolate exhibited, in addition to the 216-nt duplication, a deletion of a 40-nt repeated hairpin (RYF) motif (YF-XL-DeltaRYF). To investigate the biological significance of these 3' NCR rearrangements, YF-XL-DeltaRYF and YF-XL isolates, as well as other South American YFV isolates, were evaluated for three phenotypes: growth kinetics in cell culture, neuroinvasiveness in suckling mice, and ability to replicate and produce disseminated infections in Aedes aegypti mosquitoes. YF-XL-DeltaRYF and YF-XL isolates showed growth kinetics and neuroinvasive characteristics comparable to those of typical South American YFV isolates, and mosquito infectivity trials demonstrated that both types of 3' NCR variants were capable of replication and dissemination in a laboratory-adapted colony of A. aegypti.