Genome Characterization of Yellow Fever Virus Wild-Type Strain Asibi, Parent to Live-Attenuated 17D Vaccine, from Three Different Sources.

The yellow fever virus vaccine, 17D, was derived through the serial passage of the wild-type (WT) strain Asibi virus in mouse and chicken tissue. Since its derivation, the mechanism of attenuation of 17D virus has been investigated using three 17D substrains and WT Asibi virus. Although all three substrains of 17D have been sequenced, only one isolate of Asibi has been examined genetically and all interpretation of attenuation is based on this one isolate. Here, we sequenced the genome of Asibi virus from three different laboratories and show that the WT strain is genetically homogenous at the amino acids that distinguish Asibi from 17D vaccine virus.

Late-Relapsing Hepatitis after Yellow Fever.

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.

A randomised double-blind clinical trial of two yellow fever vaccines prepared with substrains 17DD and 17D-213/77 in children nine-23 months old

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.

Biological and phylogenetic characteristics of yellow fever virus lineages from West Africa.

The yellow fever virus (YFV), the first proven human-pathogenic virus, although isolated in 1927, is still a major public health problem, especially in West Africa where it causes outbreaks every year. Nevertheless, little is known about its genetic diversity and evolutionary dynamics, mainly due to a limited number of genomic sequences from wild virus isolates. In this study, we analyzed the phylogenetic relationships of 24 full-length genomes from YFV strains isolated between 1973 and 2005 in a sylvatic context of West Africa, including 14 isolates that had previously not been sequenced. By this, we confirmed genetic variability within one genotype by the identification of various YF lineages circulating in West Africa. Further analyses of the biological properties of these lineages revealed differential growth behavior in human liver and insect cells, correlating with the source of isolation and suggesting host adaptation. For one lineage, repeatedly isolated in a context of vertical transmission, specific characteristics in the growth behavior and unique mutations of the viral genome were observed and deserve further investigation to gain insight into mechanisms involved in YFV emergence and maintenance in nature.

Detection of a new yellow fever virus lineage within the South American genotype I in Brazil.

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.

Aplicação de PCR em tempo real para avaliar viremia em humanos e primatas não humanos, imunizados com a vacina de febre amarela cepa 17DD e o vírus recombinante 17D-DENV / Application of PCR in real time to evaluate viremia in human beings and not human primates, immunized with the vaccine of yellow fever cepa 17DD and recombinant virus 17D-DENV

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...

Analysis of two imported cases of yellow fever infection from Ivory Coast and The Gambia to Germany and Belgium.

BACKGROUND: Yellow fever remains one of the great burdens for public health in the endemic regions in Africa and South America. The under reporting of yellow fever cases in the respective regions and lack of international interest leads to an underestimation of the constant danger in these areas. Non-vaccinated travelers take a high risk without the effective protection of YFV 17D vaccination. OBJECTIVES: Two YF cases were imported to Europe in the last 4 years. We characterized two yellow fever virus (YFV) isolates from severely infected patients coming back from Africa, Ivory Coast and The Gambia, by genome sequencing and phylogenetic analysis. STUDY DESIGN: The virus infections in different organs were analyzed with pathological, immunohistological, electronmicroscopical and quantitative real-time PCR methods. RESULTS AND CONCLUSION: High virus loads in spleen and liver (2.4 x 10 (6) to 3 x 10 (7)GE/mL) demonstrated by real time PCR show massive virus replication leading to extraordinary progression of the disease in these patients. Immunohistological and electronmicroscopical analysis confirms virus particles in liver tissue. In all other organs no virus could be detected. A fast, specific and sensitive virus PCR detection is recommended for diagnostic of acute infections. The further sequence alignments show that the new isolates belong to the type II West African strain with great homology to over 40-year old YF isolates from Senegal and Ghana. The divergence observed was on average 3.3%, ranging from 0.0% to 5.0% in the coding region of Gambia 2001 strain and 2.9 %, ranging from 0.0% to 4.3% in the coding region of the Ivory C 1999 strain. Most mutations (5.0%/4.3%, respectively) occurred in the envelope protein.

Comparative phylogenies of yellow fever isolates from Peru and Brazil.

We recently reported phylogenetic evidence to support the presence of enzootic transmission foci of yellow fever virus (YFV) in Peru [Bryant et al., Emerg. Infect. Dis. (2003)]. Because the prevailing paradigm of YFV transmission in Brazil is that of 'wandering epizootics' rather than discrete enzootic foci, we have now compared the molecular phylogenies of YFV isolates from Peru and Brazil, and re-examined the question of virus mobility by mapping the spatio-temporal distribution of genetic variants from these areas. Sequences were obtained for two genomic regions from 50 strains of YFV collected between 1954 and 2000 comprising 223 codons of the structural proteins (premembrane and envelope genes, 'prM/E'), and a distal region spanning the carboxy terminus of NS5 and part of the 3' non-coding region ('EMF'). Peruvian and Brazilian isolates formed two monophyletic clades with no evidence to support recombination between lineages. Variation within both coding and non-coding regions revealed similar substitution rates and overall levels of diversity within each clade. The branching structure of the prM/E and EMF trees of Brazilian sequences showed strong agreement of intra-lineage relationships; in contrast, the EMF sequences of Peruvian isolates failed to fully support the subclade structure of the prM/E phylogeny. These phylogenies suggest that transmission cycles of YFV in Peru and Brazil may sometimes be locally maintained within specific locales, but have also on occasion become very widely dispersed.

Phylogenetic and evolutionary relationships among yellow fever virus isolates in Africa.

Previous studies with a limited number of strains have indicated that there are two genotypes of yellow fever (YF) virus in Africa, one in west Africa and the other in east and central Africa. We have examined the prM/M and a portion of the E protein for a panel of 38 wild strains of YF virus from Africa representing different countries and times of isolation. Examination of the strains revealed a more complex genetic relationship than previously reported. Overall, nucleotide substitutions varied from 0 to 25.8% and amino acid substitutions varied from 0 to 9.1%. Phylogenetic analysis using parsimony and neighbor-joining algorithms identified five distinct genotypes: central/east Africa, east Africa, Angola, west Africa I, and west Africa II. Extensive variation within genotypes was observed. Members of west African genotype II and central/east African genotype differed by 2.8% or less, while west Africa genotype I varied up to 6.8% at the nucleotide level. We speculate that the former two genotypes exist in enzootic transmission cycles, while the latter is genetically more heterogeneous due to regular human epidemics. The nucleotide sequence of the Angola genotype diverged from the others by 15.7 to 23.0% but only 0.4 to 5.6% at the amino acid level, suggesting that this genotype most likely diverged from a progenitor YF virus in east/central Africa many years ago, prior to the separation of the other east/central African strains analyzed in this study, and has evolved independently. These data demonstrate that there are multiple genotypes of YF virus in Africa and suggest independent evolution of YF virus in different areas of Africa.

Yellow fever: an update.

Yellow fever, the original viral haemorrhagic fever, was one of the most feared lethal diseases before the development of an effective vaccine. Today the disease still affects as many as 200,000 persons annually in tropical regions of Africa and South America, and poses a significant hazard to unvaccinated travellers to these areas. Yellow fever is transmitted in a cycle involving monkeys and mosquitoes, but human beings can also serve as the viraemic host for mosquito infection. Recent increases in the density and distribution of the urban mosquito vector, Aedes aegypti, as well as the rise in air travel increase the risk of introduction and spread of yellow fever to North and Central America, the Caribbean and Asia. Here I review the clinical features of the disease, its pathogenesis and pathophysiology. The disease mechanisms are poorly understood and have not been the subject of modern clinical research. Since there is no specific treatment, and management of patients with the disease is extremely problematic, the emphasis is on preventative vaccination. As a zoonosis, yellow fever cannot be eradicated, but reduction of the human disease burden is achievable through routine childhood vaccination in endemic countries, with a low cost for the benefits obtained. The biological characteristics, safety, and efficacy of live attenuated, yellow fever 17D vaccine are reviewed. New applications of yellow fever 17D virus as a vector for foreign genes hold considerable promise as a means of developing new vaccines against other viruses, and possibly against cancers.

Nucleotide sequence variation of the envelope protein gene identifies two distinct genotypes of yellow fever virus.

The evolution of yellow fever virus over 67 years was investigated by comparing the nucleotide sequences of the envelope (E) protein genes of 20 viruses isolated in Africa, the Caribbean, and South America. Uniformly weighted parsimony algorithm analysis defined two major evolutionary yellow fever virus lineages designated E genotypes I and II. E genotype I contained viruses isolated from East and Central Africa. E genotype II viruses were divided into two sublineages: IIA viruses from West Africa and IIB viruses from America, except for a 1979 virus isolated from Trinidad (TRINID79A). Unique signature patterns were identified at 111 nucleotide and 12 amino acid positions within the yellow fever virus E gene by signature pattern analysis. Yellow fever viruses from East and Central Africa contained unique signatures at 60 nucleotide and five amino acid positions, those from West Africa contained unique signatures at 25 nucleotide and two amino acid positions, and viruses from America contained such signatures at 30 nucleotide and five amino acid positions in the E gene. The dissemination of yellow fever viruses from Africa to the Americas is supported by the close genetic relatedness of genotype IIA and IIB viruses and genetic evidence of a possible second introduction of yellow fever virus from West Africa, as illustrated by the TRINID79A virus isolate. The E protein genes of American IIB yellow fever viruses had higher frequencies of amino acid substitutions than did genes of yellow fever viruses of genotypes I and IIA on the basis of comparisons with a consensus amino acid sequence for the yellow fever E gene. The great variation in the E proteins of American yellow fever virus probably results from positive selection imposed by virus interaction with different species of mosquitoes or nonhuman primates in the Americas.

Mutagenesis of conserved residues at the yellow fever virus 3/4A and 4B/5 dibasic cleavage sites: effects on cleavage efficiency and polyprotein processing.

Flavivirus proteins are produced by co- and post-translational proteolytic processing of a large polyprotein using both host- and virus-encoded enzymes. The flavivirus serine proteinase, which consists of NS2B and NS3, is responsible for cleavages of at least four dibasic sites in the nonstructural region. In this study, a number of substitutions for the conserved amino acids flanking the 3/4A and 4B/5 dibasic cleavage sites [Arg(P2)-Arg(P1) decreases Gly(P1')] were examined for their effects on yellow fever virus (YF) polyprotein processing. The substrate for these studies was a truncated YF polyprotein, called sig2A-5(356), which consists of a signal sequence fused to NS2A and extending through the first 356 amino acids of NS5. At the P1' position (Gly) of the 4B/5 site, only Ser and Ala were allowed while six other substitutions abolished cleavage. Substitutions of the 4B/5 P1 Arg residue with Lys, Gln, Asn, or His were tolerated while replacement with Glu eliminated cleavage. The 4B/5 P2 position (Arg) was found to be tolerant of substitutions with polar or hydrophobic residues which allowed varying degrees of partial cleavage. Previous studies have shown that cleavage at the 3/4A site is incomplete in YF-infected cells and that the cleavage efficiency at this site is significantly less for the sig2A-5(356) polyprotein. Replacement of the 3/4A P1 Arg residue with noncharged polar or hydrophobic residues reduced the cleavage efficiency, whereas substitutions with Glu or Pro abolished cleavage. Studies with polyproteins containing one or both of the 3/4A and 4B/5 cleavage sites blocked indicate that there is not an obligatory processing order for cleavages generating the N termini of YF NS4A, NS4B, and NS5.

Comparison of the nucleotide and deduced amino acid sequences of the envelope protein genes of the wild-type French viscerotropic strain of yellow fever virus and the live vaccine strain, French neurotropic vaccine, derived from it.

The envelope (E) protein genes of the wild-type yellow fever (YF) virus French viscerotropic virus and its' vaccine derivative, French neurotropic virus (FNV), were compared and found to differ by 13 nucleotides that coded for 8 amino acid substitutions. Comparison of the E proteins of FNV and 17D, the vaccine strain derived from wild-type strain Asibi, showed that there was no common nucleotide change or amino acid substitution between these two vaccine strains. However, changes are clustered around amino acid positions 52-56 and may represent the common vaccine epitope shared by 17D and FNV vaccine viruses. The molecular basis of any difference in neurotropism and viscerotropism of YF virus, attributable to the E protein, remains unclear.

Identification of envelope protein epitopes that are important in the attenuation process of wild-type yellow fever virus.

Monoclonal antibodies (MAbs) have been prepared against vaccine and wild-type strains of yellow fever (YF) virus, and envelope protein epitopes specific for vaccine (MAbs H5 and H6) and wild-type (MAbs S17, S18, S24, and S56) strains of YF virus have been identified. Wild-type YF virus FVV, Dakar 1279, and B4.1 were each given six passages in HeLa cells. FVV and B4.1 were attenuated for newborn mice following passage in HeLa cells, whereas Dakar 1279 was not. Examination of the envelope proteins of the viruses with 87 MAbs showed that attenuated viruses gained only the vaccine epitope recognized by MAb H5 and lost wild-type epitopes recognized by MAbs S17, S18, and S24 whereas the nonattenuated Dakar 1279 HeLa p6 virus did not gain the vaccine epitope, retained the wild-type epitopes, and showed no other physical epitope alterations. MAb neutralization-resistant (MAbr) escape variants generated by using wild-type-specific MAbs S18 and S24 were found to lose the epitopes recognized by MAbs S18 and S24 and to acquire the epitope recognized by vaccine-specific MAb H5. In addition, the MAbr variants became attenuated for mice. Thus, the data presented in this paper indicate that acquisition of vaccine epitopes and loss of wild-type epitopes on the envelope protein are directly involved in the attenuation process of YF virus and suggest that the envelope protein is one of the genes encoding determinants of YF virus pathogenicity.

Yellow fever monoclonal antibodies: type-specific and cross-reactive determinants identified by immunofluorescence.

Monoclonal antibodies directed against the envelope glycoprotein and the NV3 non-structural viral protein of yellow fever (YF) were tested by the indirect fluorescent antibody technique against a variety of YF virus strains and heterologous flaviviruses. Monoclonal antibodies directed against the envelope glycoprotein exhibited YF strain-specificity, YF type-specificity, broad group cross-reactivity, or limited subgroup reactivity (YF + Banzi or YF + Koutango + Zika + Usutu + Uganda S). Monoclonal antibodies directed against NV3 reacted either with YF + Koutango or with YF + Banzi. These findings generally correlated with the results of biological tests reported previously. Monoclonal antibodies that were type-specific to YF will be useful for the rapid specific identification of YF virus isolates and are available from the Centers for Disease Control on request.