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

ABSTRACT 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.

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Structural and Nonstructural Genes Contribute to the Genetic Diversity of RNA Viruses

mBio ◽

10.1128/mbio.01871-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 11

Author(s):

Natalie D. Collins ◽

Andrew S. Beck ◽

Steven G. Widen ◽

Thomas G. Wood ◽

Stephen Higgs ◽

...

Keyword(s):

Genetic Diversity ◽

Yellow Fever ◽

Yellow Fever Virus ◽

Rna Viruses ◽

Rna Virus ◽

Infectious Clone ◽

Vaccine Virus ◽

Wild Type ◽

Rna Dependent Rna Polymerase ◽

Replication Complex

ABSTRACT One paradigm to explain the complexity of viral RNA populations is that the low fidelity of the RNA-dependent RNA polymerase (RdRp) drives high mutation rates and consequently genetic diversity. Like most RNA viruses, wild-type yellow fever virus (YFV) replication is error-prone due to the lack of proofreading by the virus-encoded RdRp. However, there is evidence that replication of the live attenuated YF vaccine virus 17D, derived from wild-type strain Asibi, is less error-prone than wild-type RNA viruses. Recent studies comparing the genetic diversity of wild-type Asibi and 17D vaccine virus found that wild-type Asibi has the typical heterogeneous population of an RNA virus, while there is limited intra- and interpopulation variability of 17D vaccine virus. Utilizing chimeric and mutant infectious clone-derived viruses, we show that high and low genetic diversity profiles of wild-type Asibi virus and vaccine virus 17D, respectively, are multigenic. Introduction of either structural (pre-membrane and envelope) genes or NS2B or NS4B substitutions into the Asibi and 17D backbone resulted in altered variant population, nucleotide diversity, and mutation frequency compared to the parental viruses. Additionally, changes in genetic diversity of the chimeric and mutant viruses correlated with the phenotype of multiplication kinetics in human alveolar A549 cells. Overall, the paradigm that only the error-prone RdRp controls genetic diversity needs to be expanded to address the role of other genes in genetic diversity, and we hypothesize that it is the replication complex as a whole and not the RdRp alone that controls genetic diversity. IMPORTANCE With the advent of advanced sequencing technology, studies of RNA viruses have shown that genetic diversity can contribute to both attenuation and virulence and the paradigm is that this is controlled by the error-prone RNA-dependent RNA polymerase (RdRp). Since wild-type yellow fever virus (YFV) strain Asibi has genetic diversity typical of a wild-type RNA virus, while 17D virus vaccine has limited diversity, it provides a unique opportunity to investigate RNA population theory in the context of a well-characterized live attenuated vaccine. Utilizing infectious clone-derived viruses, we show that genetic diversity of RNA viruses is complex and that multiple genes, including structural genes and NS2B and NS4B genes also contribute to genetic diversity. We suggest that the replication complex as a whole, rather than only RdRp, drives genetic diversity, at least for YFV.

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Genetic Determinants of Japanese Encephalitis Virus Vaccine Strain SA14-14-2 That Govern Attenuation of Virulence in Mice

Journal of Virology ◽

10.1128/jvi.00219-15 ◽

2015 ◽

Vol 89 (12) ◽

pp. 6328-6337 ◽

Cited By ~ 27

Author(s):

Gregory D. Gromowski ◽

Cai-Yen Firestone ◽

Stephen S. Whitehead

Keyword(s):

Tissue Culture ◽

Amino Acid ◽

Japanese Encephalitis Virus ◽

Vaccine Strain ◽

Japanese Encephalitis ◽

Second Generation ◽

Virus Genome ◽

Vaccine Virus ◽

Wild Type ◽

Virulence Attenuation

ABSTRACTThe safety and efficacy of the live-attenuated Japanese encephalitis virus (JEV) SA14-14-2 vaccine are attributed to mutations that accumulated in the viral genome during its derivation. However, little is known about the contribution that is made by most of these mutations to virulence attenuation and vaccine immunogenicity. Here, we generated recombinant JEV (rJEV) strains containing JEV SA14-14-2 vaccine-specific mutations that are located in the untranslated regions (UTRs) and seven protein genes or are introduced from PCR-amplified regions of the JEV SA14-14-2 genome. The resulting mutant viruses were evaluated in tissue culture and in mice. The authentic JEV SA14-14-2 (E) protein, with amino acid substitutions L107F, E138K, I176V, T177A, E244G, Q264H, K279M, A315V, S366A, and K439R relative to the wild-type rJEV clone, was essential and sufficient for complete attenuation of neurovirulence. Individually, the nucleotide substitution T39A in the 5′ UTR (5′-UTR-T39A), the capsid (C) protein amino acid substitution L66S (C-L66S), and the complete NS1/2A genome region containing 10 mutations each significantly reduced virus neuroinvasion but not neurovirulence. The levels of peripheral virulence attenuation imposed by the 5′-UTR-T39A and C-L66S mutations, individually, were somewhat mitigated in combination with other vaccine strain-specific mutations, which might be compensatory, and together did not affect immunogenicity. However, a marked reduction in immunogenicity was observed with the addition of the NS1/2A and NS5 vaccine virus genome regions. These results suggest that a second-generation recombinant vaccine can be rationally engineered to maximize levels of immunogenicity without compromising safety.IMPORTANCEThe live-attenuated JEV SA14-14-2 vaccine has been vital for controlling the incidence of disease caused by JEV, particularly in rural areas of Asia where it is endemic. The vaccine was developed >25 years ago by passaging wild-type JEV strain SA14 in tissue cultures and rodents, with intermittent tissue culture plaque purifications, to produce a virus clone that had adequate levels of attenuation and immunogenicity. The vaccine and parent virus sequences were later compared, and mutations were identified throughout the vaccine virus genome, but their contributions to attenuation were never fully elucidated. Here, using reverse genetics, we comprehensively defined the impact of JEV SA14-14-2 mutations on attenuation of virulence and immunogenicity in mice. These results are relevant for quality control of new lots of the current live-attenuated vaccine and provide insight for the rational design of second-generation, live-attenuated, recombinant JEV vaccine candidates.

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

Viruses ◽

10.3390/v13071383 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1383

Author(s):

Emily H. Davis ◽

Jill K. Thompson ◽

Steven G. Widen ◽

Alan D. T. Barrett

Keyword(s):

Yellow Fever ◽

Wild Type Strain ◽

Yellow Fever Virus ◽

Serial Passage ◽

Vaccine Virus ◽

Fever Virus ◽

Wild Type ◽

Chicken Tissue ◽

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.

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

Journal of General Virology ◽

10.1099/vir.0.031617-0 ◽

2011 ◽

Vol 92 (10) ◽

pp. 2262-2271 ◽

Cited By ~ 11

Author(s):

Sara E. Woodson ◽

Michael R. Holbrook

Keyword(s):

Yellow Fever ◽

Vaccine Strain ◽

Host Response ◽

Yellow Fever Virus ◽

Vaccine Virus ◽

Fever Virus ◽

Productive Infection ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type

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.

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Japanese encephalitis virus live attenuated vaccine strains display altered immunogenicity, virulence and genetic diversity

npj Vaccines ◽

10.1038/s41541-021-00371-y ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Emily H. Davis ◽

Andrew S. Beck ◽

Li Li ◽

Mellodee M. White ◽

Marianne Banks Greenberg ◽

...

Keyword(s):

Genetic Diversity ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Serial Passage ◽

Encephalitis Virus ◽

Wild Type ◽

Live Attenuated Vaccine ◽

Replication Complex ◽

Generation Sequencing

AbstractJapanese encephalitis virus (JEV) is the etiological agent of Japanese encephalitis (JE). The most commonly used vaccine used to prevent JE is the live-attenuated strain SA14-14-2, which was generated by serial passage of the wild-type (WT) JEV strain SA14. Two other vaccine candidates, SA14-5-3 and SA14-2-8 were derived from SA14. Both were shown to be attenuated but lacked sufficient immunogenicity to be considered effective vaccines. To better contrast the SA14-14-2 vaccine with its less-immunogenic counterparts, genetic diversity, ribavirin sensitivity, mouse virulence and mouse immunogenicity of the three vaccines were investigated. Next generation sequencing demonstrated that SA14-14-2 was significantly more diverse than both SA14-5-3 and SA14-2-8, and was slightly less diverse than WT SA14. Notably, WT SA14 had unpredictable levels of diversity across its genome whereas SA14-14-2 is highly diverse, but genetic diversity is not random, rather the virus only tolerates variability at certain residues. Using Ribavirin sensitivity in vitro, it was found that SA14-14-2 has a lower fidelity replication complex compared to SA14-5-3 and SA14-2-8. Mouse virulence studies showed that SA14-2-8 was the most virulent of the three vaccine strains while SA14-14-2 had the most favorable combination of safety (virulence) and immunogenicity for all vaccines tested. SA14-14-2 contains genetic diversity and sensitivity to the antiviral Ribavirin similar to WT parent SA14, and this genetic diversity likely explains the (1) differences in genomic sequences reported for SA14-14-2 and (2) the encoding of major attenuation determinants by the viral E protein.

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Ever-increasing viral diversity associated with the red imported fire ant Solenopsis invicta (Formicidae: Hymenoptera)

Virology Journal ◽

10.1186/s12985-020-01469-w ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

César Augusto Diniz Xavier ◽

Margaret Louise Allen ◽

Anna Elizabeth Whitfield

Keyword(s):

Solenopsis Invicta ◽

De Novo ◽

Rna Virus ◽

Housekeeping Genes ◽

Virus Genome ◽

Single Strand ◽

Viral Diversity ◽

Red Imported Fire Ant ◽

Sequence Comparisons ◽

Data Set

Abstract Background Advances in sequencing and analysis tools have facilitated discovery of many new viruses from invertebrates, including ants. Solenopsis invicta is an invasive ant that has quickly spread worldwide causing significant ecological and economic impacts. Its virome has begun to be characterized pertaining to potential use of viruses as natural enemies. Although the S. invicta virome is the best characterized among ants, most studies have been performed in its native range, with less information from invaded areas. Methods Using a metatranscriptome approach, we further identified and molecularly characterized virus sequences associated with S. invicta, in two introduced areas, U.S and Taiwan. The data set used here was obtained from different stages (larvae, pupa, and adults) of S. invicta life cycle. Publicly available RNA sequences from GenBank’s Sequence Read Archive were downloaded and de novo assembled using CLC Genomics Workbench 20.0.1. Contigs were compared against the non-redundant protein sequences and those showing similarity to viral sequences were further analyzed. Results We characterized five putative new viruses associated with S. invicta transcriptomes. Sequence comparisons revealed extensive divergence across ORFs and genomic regions with most of them sharing less than 40% amino acid identity with those closest homologous sequences previously characterized. The first negative-sense single-stranded RNA virus genomic sequences included in the orders Bunyavirales and Mononegavirales are reported. In addition, two positive single-strand virus genome sequences and one single strand DNA virus genome sequence were also identified. While the presence of a putative tenuivirus associated with S. invicta was previously suggested to be a contamination, here we characterized and present strong evidence that Solenopsis invicta virus 14 (SINV-14) is a tenui-like virus that has a long-term association with the ant. Furthermore, based on virus sequence abundance compared to housekeeping genes, phylogenetic relationships, and completeness of viral coding sequences, our results suggest that four of five virus sequences reported, those being SINV-14, SINV-15, SINV-16 and SINV-17, may be associated to viruses actively replicating in the ant S. invicta. Conclusions The present study expands our knowledge about viral diversity associated with S. invicta in introduced areas with potential to be used as biological control agents, which will require further biological characterization.

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Role of the Three Polyoma Virus Early Proteins in Tumorigenesis

Molecular and Cellular Biology ◽

10.1128/mcb.3.8.1451 ◽

1983 ◽

Vol 3 (8) ◽

pp. 1451-1459 ◽

Cited By ~ 29

Author(s):

Claude Asselin ◽

Celine Gelinas ◽

Marcel Bastin

Keyword(s):

Cultured Cells ◽

Virus Genome ◽

T Antigen ◽

Polyoma Virus ◽

Transformed Cells ◽

Newborn Rats ◽

Wild Type ◽

Complementary Function ◽

Small T Antigen ◽

Middle T Antigen

A modified polyoma virus genome which can encode the middle T protein but not the large or small T proteins transforms rat cells in culture with an efficiency about 20% that of the wild-type genome. Although middle T-transformed cells grow as tumors when transplanted into nude mice or syngeneic rats, the middle T gene alone is totally inactive when used in a more stringent and rigorous assay for tumorigenicity such as the injection of DNA into newborn rats. Thus, functions other than those expressed by middle T antigen are required for the elaboration of all the properties associated with tumorigenesis. To assess whether a complementary function could be exerted by the large or the small T antigen, we constructed plasmids containing two modified early regions which independently encoded middle T and one of the two other proteins. Both recombinants were tumorigenic in newborn rats. Cell lines derived by transfer of these plasmids under no special selective conditions did not acquire the property of growth in low-serum medium but exhibited the same tumorigenic properties as wild-type polyoma DNA-transformed cells. Furthermore, a recombinant which encoded the middle and small T antigens, but not the large T antigen, was tumorigenic in newborn rats. Although the small T antigen provides a complementary function for tumorigenicity, it cannot complement the middle T antigen for an efficient induction of transformation of cultured cells. This suggests that the complementary function exerted by the small T antigen is different from that of the N-terminal fragment of the large T protein.

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Liposomal Systems as Nanocarriers for the Antiviral Agent Ivermectin

International Journal of Biomaterials ◽

10.1155/2016/8043983 ◽

2016 ◽

Vol 2016 ◽

pp. 1-15 ◽

Cited By ~ 11

Author(s):

Romina Croci ◽

Elisabetta Bottaro ◽

Kitti Wing Ki Chan ◽

Satoru Watanabe ◽

Margherita Pezzullo ◽

...

Keyword(s):

Yellow Fever ◽

Rna Virus ◽

Multiorgan Failure ◽

Antiviral Agent ◽

Drug Carriers ◽

Virus Infections ◽

Starting Point ◽

Significant Public Health ◽

High Cytotoxicity

RNA virus infections can lead to the onset of severe diseases such as fever with haemorrhage, multiorgan failure, and mortality. The emergence and reemergence of RNA viruses continue to pose a significant public health threat worldwide with particular attention to the increasing incidence of flaviviruses, among others Dengue, West Nile Virus, and Yellow Fever viruses. Development of new and potent antivirals is thus urgently needed. Ivermectin, an already known antihelminthic drug, has shown potent effectsin vitroonFlavivirushelicase, with EC50values in the subnanomolar range for Yellow Fever and submicromolar EC50for Dengue Fever, Japanese encephalitis, and tick-borne encephalitis viruses. However ivermectin is hampered in its application by pharmacokinetic problems (little solubility and high cytotoxicity). To overcome such problems we engineered different compositions of liposomes as ivermectin carriers characterizing and testing them on several cell lines for cytotoxicity. The engineered liposomes were less cytotoxic than ivermectin alone and they showed a significant increase of the antiviral activity in all the Dengue stains tested (1, 2, and S221). In the current study ivermectin is confirmed to be an effective potential antiviral and liposomes, as drug carriers, are shown to modulate the drug activity. All together the results represent a promising starting point for future improvement of ivermectin as antiviral and its delivery.

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