Asibi Virus Research Articles

Genetic Diversity Does Not Contribute to Attenuation of HeLa Passaged Wild-Type Yellow Fever Virus Strain French Viscerotropic Virus.

The disease yellow fever was prevented by two live attenuated vaccines, strains 17D and French neurotropic vaccine (FNV), derived by serial passage of wild-type (WT) strains Asibi and French Viscerotropic virus (FVV), respectively. Both 17D and FNV displayed decreased genetic diversity and resistance to the antiviral Ribavirin compared to their WT parental strains, which are thought to contribute to their attenuated phenotypes. Subsequent studies found that only a few passages of WT strain FVV in HeLa cells resulted in an attenuated virus. In the current study, the genome sequence of FVV following five passages in HeLa cells (FVV HeLa p5) was determined through Next Generation Sequencing (NGS) with the aim to investigate the molecular basis of viral attenuation. It was found that WT FVV and FVV HeLa p5 virus differed by five amino acid substitutions: E-D155A, E-K331R, E-I412V, NS2A-T105A, and NS4B-V98I. Surprisingly, the genetic diversity and Ribavirin resistance of the FVV HeLa p5 virus were not statistically different to WT parent FVV. These findings suggest that while FVV HeLa p5 is attenuated, this is not dependent on a high-fidelity replication complex, characterized by reduced genetic diversity or increased Ribavirin stability, as seen with FNV and 17D vaccines.

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.

Attenuation of Live-Attenuated Yellow Fever 17D Vaccine Virus Is Localized to a High-Fidelity Replication Complex.

The molecular basis of attenuation for live-attenuated vaccines is poorly understood. The yellow fever (YF) 17D vaccine virus was derived from the wild-type, parental strain Asibi virus by serial passage in chicken tissue and has proven to be a very safe and efficacious vaccine. We have previously shown that wild-type Asibi is a typical RNA virus with high genetic diversity, while the 17D vaccine virus has very little genetic diversity. To investigate this further, we treated Asibi and 17D viruses with ribavirin, a GTP analog with strong antiviral activity that increases levels of mutations in the viral genome. As expected, ribavirin treatment introduced mutations into the Asibi virus genome at a very high frequency and decreased viral infectivity while, in contrast, the 17D vaccine virus was resistant to ribavirin, as treatment with the antiviral introduced very few mutations into the genome, and viral infectivity was not lost. The results were confirmed for another YF wild-type parental and vaccine pair, a wild-type French viscerotropic virus and French neurotropic vaccine. Using recombinant Asibi and 17D viruses, ribavirin sensitivity was located to viral nonstructural genes. Thus, two live-attenuated YF vaccine viruses are genetically stable even under intense mutagenic pressure, suggesting that attenuation of live-attenuated YF vaccines is due, at least in part, to fidelity of the replication complex resulting in high genetic stability.IMPORTANCE Live-attenuated viral vaccines are highly safe and efficacious but represent complex and often multigenic attenuation mechanisms. Most of these vaccines have been generated empirically by serial passaging of a wild-type (WT) virus in cell culture. One of the safest and most effective live-attenuated vaccines is yellow fever (YF) virus strain 17D, which has been used for over 80 years to control YF disease. The availability of the WT parental strain of 17D, Asibi virus, and large quantities of clinical data showing the effectiveness of the 17D vaccine make this WT parent/vaccine pair an excellent model for investigating RNA virus attenuation. Here, we investigate a mechanism of 17D attenuation and show that the vaccine virus is resistant to the antiviral compound ribavirin. The findings suggest that attenuation is in part due to a low probability of reversion or mutation of the vaccine virus genome to WT, thus maintaining a stable genotype despite external pressures.

Molecular determinants of Yellow Fever Virus pathogenicity in Syrian Golden Hamsters: one mutation away from virulence

Yellow fever virus (Flavivirus genus) is an arthropod-borne pathogen, which can infect humans, causing a severe viscerotropic disease with a high mortality rate. Adapted viral strains allow the reproduction of yellow fever disease in hamsters with features similar to the human disease. Here, we used the Infectious Subgenomic Amplicons reverse genetics method to produce an equivalent to the hamster-virulent strain, Yellow Fever Ap7, by introducing a set of four synonymous and six nonsynonymous mutations into a single subgenomic amplicon, derived from the sequence of the Asibi strain. The resulting strain, Yellow Fever Ap7M, induced a disease similar to that described for Ap7 in terms of symptoms, weight evolution, viral loads in the liver and lethality. Using the same methodology, we produced mutant strains derived from either Ap7M or Asibi viruses and investigated the role of each of Ap7M nonsynonymous mutations in its in vivo phenotype. This allowed identifying key components of the virulence mechanism in hamsters. In Ap7M virus, the reversion of either E/Q27H or E/D155A mutations led to an important reduction of both virulence and in vivo replicative fitness. In addition, the introduction of the single D155A Ap7M mutation within the E protein of the Asibi virus was sufficient to drastically modify its phenotype in hamsters toward both a greater replication efficiency and virulence. Finally, inspection of the Asibi strain E protein structure combined to in vivo testing revealed the importance of an exposed α-helix in domain I, containing residues 154 and 155, for Ap7M virulence in hamsters.

Deep sequencing of the yellow fever vaccine strain 17D reveals low diversity compared to wild-type parent Asibi virus and population stability for primary and secondary seed lots of 17D vaccine

Deep sequencing of the yellow fever vaccine strain 17D reveals low diversity compared to wild-type parent Asibi virus and population stability for primary and secondary seed lots of 17D vaccine

Infection of hepatocytes with 17-D vaccine-strain yellow fever virus induces a strong pro-inflammatory host response

Yellow fever virus (YFV) causes serious disease in endemic areas of South America and Africa, even though a very well tolerated vaccine is available. YFV primarily targets the liver where as many as 80 % of hepatocytes may be involved during infection. The objective of this project was to compare and contrast the cytokine response from hepatocytes infected with either wild-type (Asibi) or vaccine (17-D-204) strains of YFV, with the goal of identifying responses that might be correlated with disease severity or vaccine efficacy. We report here that PH5CH8 hepatocytes support a productive infection with both wild-type and vaccine-strain YFV. Infection with either virus resulted in elevated expression of several pro- and anti-inflammatory cytokines [interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10 and tumour necrosis factor-α) with a corresponding increase in transcription. Hepatocytes infected with vaccine virus had a more profound response than did cells infected with wild-type virus. Pre-stimulation of hepatocytes with IL-6 resulted in reduced viral titres, elevated concentrations of cytokines released from Asibi virus-infected cells and improved cell viability in cells infected with 17-D virus. Data reported here suggest that 17-D virus stimulates an appropriate antiviral inflammatory response in hepatocytes, while Asibi virus can attenuate the host response. These data identify potential mechanisms that are associated with increased virulence in wild-type virus infections and also provide clues towards potential immune-response limitations that may be associated with vaccine-related adverse events.

Phenotypic and molecular characterization of a non-lethal, hamster-viscerotropic strain of yellow fever virus

Viscerotropic yellow fever virus (YFV) infection occurs primarily in humans and non-human primates. Lack of an appropriate small animal model of viscerotropic YFV infection has been a major deterrent to molecular studies of viscerotropism. A hamster model of viscerotropic YFV infection has recently been described; however, these studies have focused on hamster-viscerotropic strains of YFV (including Asibi hamster P7 virus) that caused outward clinical signs of infection and mortality. In order to map more closely the molecular determinants of viscerotropism in the hamster model, a second sequential series of seven liver-to-liver passages of Asibi virus was undertaken through hamsters to generate Asibi P7b virus. Asibi hamster P7b virus did not cause clinically detectable signs of YFV infection; however, high quantities of circulating virus were isolated from the serum, and microscopic evaluation of the liver and spleen demonstrated histopathological lesions consistent with YFV infection. The genomic sequence of Asibi P7b virus was determined and compared to wild-type Asibi virus and the lethal, hamster-viscerotropic Asibi P7 virus and found to differ by only two amino acids in the envelope protein, E-98 and E-331.

Interaction of yellow fever virus French neurotropic vaccine strain with monkey brain: characterization of monkey brain membrane receptor escape variants.

Binding of yellow fever virus wild-type strains Asibi and French viscerotropic virus and vaccine strains 17D and FNV to monkey brain and monkey liver cell membrane receptor preparations (MRPs) was investigated. Only FNV bound to monkey brain MRPs, while French viscerotropic virus, Asibi, and FNV all bound to monkey liver MRPs. Four monkey brain and two mouse brain MRP escape (MRP(R)) variants of FNV were selected at pH 7.6 and 6.0. Three monkey brain MRP(R) variants selected at pH 7.6 each had only one amino acid substitution in the envelope (E) protein in domain II (E-237, E-260, or E274) and were significantly attenuated in mice following intracerebral inoculation. Two of the variants were tested in monkeys and retained parental neurotropism following intracerebral inoculation at the dose tested. We speculate that this region of domain II is involved in binding of FNV E protein to monkey brain and is, in part, responsible for the enhanced neurotropism of FNV for monkeys. A monkey brain MRP(R) variant selected at pH 6.0 and two mouse brain MRP(R) variants selected at pH 7.6 were less attenuated in mice, and each had an amino acid substitution in the transmembrane region of the E protein (E-457 or E-458).

Molecular and Biological Changes Associated with HeLa Cell Attenuation of Wild-Type Yellow Fever Virus

Six passages of the mosquito-borne flavivirus yellow fever (YF) wild-type strain Asibi in HeLa cells attenuated the virus for monkeys and newborn mice and resulted in loss of mosquito competence. Attenuation after the passage in HeLa cells was not unique to YF virus strain Asibi as demonstrated by the HeLa passage attenuation of wild-type YF virus strain French viscerotropic virus and YF vaccine virus 17D-204 for newborn mice. In contrast, wild-type strain Dakar 1279 and the French neurotropic vaccine virus remained virulent for newborn mice after six passages in HeLa cells. Thus not all strains of YF virus can be attenuated by passage in HeLa cells. Attenuation of YF virus strains Asibi and French viscerotropic virus was accompanied by alterations in the antigenic and biological properties of the viruses, including changes to envelope protein epitopes. Attenuation for newborn mice was coincidental with the acquisition by the HeLa-passaged viruses of the vaccine-specific envelope protein epitope recognized by monoclonal antibody H5. This suggests that this conformational change may play a role in the attenuation process. Wild-type Dakar 1279, which remained virulent for newborn mice after passage in HeLa cells, retained its wild-type antigenic character. The genome of Asibi HeLa p6 virus differed from wild-type Asibi virus by 29 nucleotides that encoded 10 amino acid substitutions: 5 in the envelope protein, 1 in NS2A, 3 in NS4B, and 1 in NS5. The substitution at NS4B-95 is seen in three different attenuation processes of wild-type YF virus, leading us to speculate that it is involved in the attenuation of virulence of wild-type strain Asibi.

Mutation in a 17D-204 Vaccine Substrain-Specific Envelope Protein Epitope Alters the Pathogenesis of Yellow Fever Virus in Mice

The heterogeneous nature of the yellow fever (YF) 17D-204 vaccine virus population was exploited in this study to isolate virus variants able to escape neutralization by the 17D-204 vaccine-specific MAb 864. The conformational change on the virus surface that resulted in the loss of the MAb 864-defined epitope was effected in each variant by a single amino acid mutation in the envelope (E) protein at either position E-305 or E-325. Interestingly, both positions were mutated during attenuation of the 17D-204 vaccine substrain from the wildtype Asibi strain. The mutations in several of the variants represented reversion to the wildtype Asibi virus sequence consistent with loss of a 17D-204 substrain-specific epitope. The majority of the variant viruses were shown to have altered mouse neurovirulence phenotypes, ranging from complete avirulence through to increased virulence. The avirulent variants are the first flavivirus MAb-neutralization-resistant variants to be attenuated for neurovirulence in the adult mouse model. Overall, the results indicate that the E protein epitope recognized by MAb 864 defines a functionally important region that encodes major molecular determinants of YF virus pathogenesisin vivo.

Effect of administration of sodium aurothiomalate on the virulence of yellow fever viruses in adult mice

Administration of sodium aurothiomalate (SATM) to adult mice results in a reduction of their average survival time (AST) following intracerebral challenge with the wild-type strains, Asibi and French viscerotropic virus (FVV), of yellow fever (YF) virus. Most attenuated 17D YF vaccines, derived by passage of the wild-type Asibi strain in chick tissue, showed no reduction in AST following intracerebral challenge and administration of SATM. In contrast, challenge with the majority of live attenuated French neurotropic vaccines, derived by passage of FVV in mouse brain, still resulted in SATM reducing the AST of mice. SATM also changed some YF viruses from non-lethal to lethal following intraperitoneal challenge and negated the ability of a monoclonal antibody to elicit passive protection of mice challenged intracerebrally with YF virus.

Identification of Monoclonal Antibodies that Distinguish Between 17D-204 and other Strains of Yellow Fever Virus

Eight monoclonal antibodies (MAbs) prepared against the flaviviruses Saint Louis encephalitis, dengue 2 and dengue 3 viruses all recognized epitopes on the envelope protein of the prototype flavivirus, yellow fever (YF) virus. Three of these MAbs with flavivirus group-common specificity and two MAbs with a flavivirus-subgroup specificity were found to distinguish wild-type YF viruses from YF 17D-204 vaccine virus, but not from the closely related 17DD vaccine virus, nor from the French neurotropic vaccine virus. This pattern of reactivity was seen only with viruses grown in Aedes albopictus C6/36 cells and not with viruses grown in vertebrate cells (SW13 and Vero cells), where all five MAbs recognized epitopes on both wild-type and 17D-204 viruses. Examination of adult A. aegypti mosquitoes infected with the same YF viruses as above gave a different pattern of results to those in C6/36 cells. Thus, epitope expression differs between mammalian and arthropod cells and between arthropod cells in vitro and in vivo. Neutralization tests showed that all five MAbs would neutralize wild-type Asibi virus grown in SW13 cells, but not Asibi virus grown in C6/36 cells, nor 17D-204 vaccine virus grown in either cell type. Therefore, it is concluded that when YF virus is grown in mosquito cells, wild-type virus is antigenically and biologically distinct from the 17D-204 vaccine virus.

Use of a Monoclonal Antibody Specific for Wild-type Yellow Fever Virus to Identify a Wild-type Antigenic Variant in 17D Vaccine Pools

Monoclonal antibodies (MAbs) against the Asibi wild-type strain of yellow fever (YF) virus were prepared and characterized. One of the MAbs (designated MAb 117) was shown, by cross-immunofluorescence tests with flaviviruses, to be specific for wildtype YF virus. This MAb was used in indirect immunofluorescence tests to identify wild-type antigenic variants in several different YF vaccine pools. Simultaneously, a vaccine-specific MAb prepared previously (MAb 864) was used to identify YF strain 17D vaccine type variants in the wild-type Asibi virus preparation. One variant, isolated by plaque purification from a 17D vaccine pool, possessed the wild-type epitope and was neurovirulent in infant mice whereas other variants, lacking the wild-type epitope but with vaccine-specific epitopes (identified by MAb 411), were avirulent in infant mice. Avirulent variants were able to infect mice and induce antibody. Virus-specific antigen was still detected in the brains of these mice 4 weeks after inoculation, suggesting that persistent infections were developing. These results to the potential risk of selection of wild-type variants in YF vaccine preparations. They also point to the potential risk of selection of wild-type variants in YF vaccine preparations and re-emphasize the need for modernization of techniques and more effective control measures to be taken during the production of YF vaccine.

Topographical analysis of epitope relationships on the envelope glycoprotein of yellow fever 17D vaccine and the wild type asibi parent virus

Monoclonal antibodies (MCA), with defined molecular specificity, were used in a competition binding enzyme-linked immunosorbent assay (ELISA) to locate the relative positions of the epitopes on the envelope glycoprotein of yellow fever 17D vaccine virus and its wild type parent virus, Asibi (AS). Five topographically distinct antigenic domains were defined on the E glycoprotein of the 17D vaccine. Three of these (A, B, and C) were represented by one MCA each, a fourth (D) was represented by two MCA, and a fifth domain (E) comprised a major cluster of at least five overlapping epitopes. Asibi virus also possessed domain E which is proposed to be a conserved antigenic region within the envelope glycoprotein of all flaviviruses. Domains A and C were not represented on Asibi virus and one epitope, situated proximal to the E domain, showed structural alterations in physical overlap. Functional activities were assigned to physically mapped epitopes by haemagglutination inhibition (HAI), virus neutralisation (N), and passive protection in mice. The HAI and N functions were not necessarily linked but only MCA with N activity were able to protect mice passively against lethal infection. All domains demonstrated a heterogeneous range of biological properties dependent upon the virus strain rather than the epitope.

Ontogeny of yellow fever 17D vaccine: RNA oligonucleotide fingerprint and monoclonal antibody analyses of vaccines produced world-wide.

Yellow fever 17D vaccines are currently manufactured with approval of the World Health Organization (WHO) in 11 countries. These vaccines have proven highly efficacious and safe. Nevertheless, they have not been fully characterized genetically, a problem for future standardization and modernization of vaccine manufacture now being proposed by WHO. Vaccines in use are derived from two distinct substrains (17D-204 and 17DD) which represent independently maintained passage series from original 17D. In this study, all 17D vaccines produced world-wide were characterized by RNA oligonucleotide fingerprinting. Forty-two large oligonucleotides were compared, and differences from an arbitrarily selected reference strain (produced by Connaught Laboratories in the U.S.A.) were determined. With one exception (vaccine produced in South Africa), fingerprints of vaccines derived from substrain 17D-204 were identical. The South African primary seed differed in position of one oligonucleotide, reflecting a charge shift due to a single base change. This difference occurred within one egg passage; a further change in the South African vaccine occurred within one or two passages from primary seed. No antigenic differences between 17D-204-derived vaccines (including South Africa) were demonstrated by neutralization tests using monoclonal antibody. Vaccines derived from the 17DD substrain consistently differed from 17D-204 vaccines in the absence of one oligonucleotide (No. 37). This change probably occurred during 40 additional egg passages in development of the 17DD vaccines. A clear antigenic difference was shown between 17D-204 and 17DD substrain vaccines using monoclonal antibody. 17DD vaccines showed minor genotypic differences, suggesting a higher degree of genetic instability than 17D-204 vaccines. No oligonucleotide fingerprint differences were found between avian leukosis virus (ALV)-free and ALV-contaminated vaccines. No definite genomic correlate of neurovirulence was defined by fingerprinting strains with a history of encephalitic complications in man or of failure to pass monkey neurovirulence tests. Parent Asibi virus showed several oligonucleotide differences and was serologically distinct from 17D vaccine.

Suscetibilidade de camondongos ao virus amarílico administrado por vias extraneurais

1. Virus neurotrópico Francês, Asibi e de vacina (17D) infectaram camondongos jovens, quando instilados nas narinas, matando-os com encefalite ou desenvolvendo imunidade. 2. Virus neurotrópico Francês e Asibi (e talvez o virus de vacina) quando instilados no ouvido de camondongos jovens, podem infectá-los produzindo o desenvolvimento de imunidade. 3. Após instilação na córnea escarificada de camondongos jovens, virus neurotrópicos Francês, Asibi e, talvez de vacina, podem produzir infecção. Com a córnea íntegra, no entando, não foi possivel infectar camondongos. 4. virus neurotrópicos Francês, Asibi, de vacina, Asibi Egg I e Egg III introduzidos no estômago, podem infectar camondongos jovens, matando-os com encefalite ou imunizando-os. 5. É possivel infectar camondongos de 0-6 dias de idade por simples deposição de virus ou esfregando a suspensão na pele íntegra e escarificada.

Treatment of Experimental Yellow Fever Encephalitis with Specific Immune Serum

The course and outcome of experimental yellow fever in Macacus rhesus when specific immune serum was injected into the animals at various intervals after the administration of the virus, has been studied by Davis (1).Penna (2) has described the changes which he observed in a viscerotropic (the Asibi) strain of yellow fever virus as a result of serial passage through the central nervous tissues of rhesus monkeys that were protected against serious visceral damage by the inoculation of specific immune serum. After about forty such passages the virus was so modified as to be primarily neurotropic.In the study here reported observations were made upon the course and outcome of yellow fever encephalitis in M. rhesus that received immune serum via the cisterna magna—after the removal of some cerebrospinal fluid—at various intervals before the intracerebral inoculation of the modified Asibi virus with which Penna was working.