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

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

mBio ◽

10.1128/mbio.02294-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 2

Author(s):

Emily H. Davis ◽

Andrew S. Beck ◽

Ashley E. Strother ◽

Jill K. Thompson ◽

Steven G. Widen ◽

...

Keyword(s):

Genetic Diversity ◽

Yellow Fever ◽

Parental Strain ◽

Rna Virus ◽

Virus Genome ◽

Vaccine Virus ◽

Viral Infectivity ◽

Wild Type ◽

Replication Complex ◽

Live Attenuated Vaccines

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

Journal of Virology ◽

10.1128/jvi.66.7.4265-4270.1992 ◽

1992 ◽

Vol 66 (7) ◽

pp. 4265-4270 ◽

Cited By ~ 18

Author(s):

B K Sil ◽

L M Dunster ◽

T N Ledger ◽

M R Wills ◽

P D Minor ◽

...

Keyword(s):

Yellow Fever ◽

Envelope Protein ◽

Yellow Fever Virus ◽

Fever Virus ◽

Wild Type

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Pluripotency of Wolbachia against Arboviruses: the case of yellow fever

Gates Open Research ◽

10.12688/gatesopenres.12903.2 ◽

2019 ◽

Vol 3 ◽

pp. 161 ◽

Cited By ~ 7

Author(s):

Marcele Neves Rocha ◽

Myrian Morato Duarte ◽

Simone Brutman Mansur ◽

Bianca Daoud Mafra e Silva ◽

Thiago Nunes Pereira ◽

...

Keyword(s):

Yellow Fever ◽

Yellow Fever Virus ◽

Oral Feeding ◽

Risk Of Infection ◽

Wild Type ◽

Transmission Potential ◽

Mosquito Saliva ◽

Tropical Cities ◽

Complementary Strategy ◽

Viral Isolates

Background: Yellow fever outbreaks have re-emerged in Brazil during 2016-18, with mortality rates up to 30%. Although urban transmission has not been reported since 1942, the risk of re-urbanization of yellow fever is significant, as Aedes aegypti is present in most tropical and sub-tropical cities in the World and still remains the main vector of urban YFV. Although the YFV vaccine is safe and effective, it does not always reach populations at greatest risk of infection and there is an acknowledged global shortage of vaccine supply. The introgression of Wolbachia bacteria into Ae. aegypti mosquito populations is being trialed in several countries (www.worldmosquito.org) as a biocontrol method against dengue, Zika and chikungunya. Here, we studied the ability of Wolbachia to reduce the transmission potential of Ae. aegypti mosquitoes for Yellow fever virus (YFV). Methods: Two recently isolated YFV (primate and human) were used to challenge field-derived wild-type and Wolbachia-infected (wMel +) Ae. aegypti mosquitoes. The YFV infection status was followed for 7, 14 and 21 days post-oral feeding (dpf). The YFV transmission potential of mosquitoes was evaluated via nano-injection of saliva into uninfected mosquitoes or by inoculation in mice. Results: We found that Wolbachia was able to significantly reduce the prevalence of mosquitoes with YFV infected heads and thoraces for both viral isolates. Furthermore, analyses of mosquito saliva, through indirect injection into naïve mosquitoes or via interferon-deficient mouse model, indicated Wolbachia was associated with profound reduction in the YFV transmission potential of mosquitoes (14dpf). Conclusions: Our results suggest that Wolbachia introgression could be used as a complementary strategy for prevention of urban yellow fever transmission, along with the human vaccination program.

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Oligomerization of the Vesicular Stomatitis Virus Phosphoprotein Is Dispensable for mRNA Synthesis but Facilitates RNA Replication

Journal of Virology ◽

10.1128/jvi.00115-20 ◽

2020 ◽

Vol 94 (13) ◽

Cited By ~ 2

Author(s):

Louis-Marie Bloyet ◽

Benjamin Morin ◽

Vesna Brusic ◽

Erica Gardner ◽

Robin A. Ross ◽

...

Keyword(s):

Vesicular Stomatitis Virus ◽

Rna Viruses ◽

Rna Virus ◽

Rna Replication ◽

Binding Domain ◽

Genome Replication ◽

Mrna Synthesis ◽

Wild Type ◽

Vesicular Stomatitis ◽

Oligomerization Domain

ABSTRACT Nonsegmented negative-strand (NNS) RNA viruses possess a ribonucleoprotein template in which the genomic RNA is sequestered within a homopolymer of nucleocapsid protein (N). The viral RNA-dependent RNA polymerase (RdRP) resides within an approximately 250-kDa large protein (L), along with unconventional mRNA capping enzymes: a GDP:polyribonucleotidyltransferase (PRNT) and a dual-specificity mRNA cap methylase (MT). To gain access to the N-RNA template and orchestrate the LRdRP, LPRNT, and LMT, an oligomeric phosphoprotein (P) is required. Vesicular stomatitis virus (VSV) P is dimeric with an oligomerization domain (OD) separating two largely disordered regions followed by a globular C-terminal domain that binds the template. P is also responsible for bringing new N protomers onto the nascent RNA during genome replication. We show VSV P lacking the OD (PΔOD) is monomeric but is indistinguishable from wild-type P in supporting mRNA transcription in vitro. Recombinant virus VSV-PΔOD exhibits a pronounced kinetic delay in progeny virus production. Fluorescence recovery after photobleaching demonstrates that PΔOD diffuses 6-fold more rapidly than the wild type within viral replication compartments. A well-characterized defective interfering particle of VSV (DI-T) that is only competent for RNA replication requires significantly higher levels of N to drive RNA replication in the presence of PΔOD. We conclude P oligomerization is not required for mRNA synthesis but enhances genome replication by facilitating RNA encapsidation. IMPORTANCE All NNS RNA viruses, including the human pathogens rabies, measles, respiratory syncytial virus, Nipah, and Ebola, possess an essential L-protein cofactor, required to access the N-RNA template and coordinate the various enzymatic activities of L. The polymerase cofactors share a similar modular organization of a soluble N-binding domain and a template-binding domain separated by a central oligomerization domain. Using a prototype of NNS RNA virus gene expression, vesicular stomatitis virus (VSV), we determined the importance of P oligomerization. We find that oligomerization of VSV P is not required for any step of viral mRNA synthesis but is required for efficient RNA replication. We present evidence that this likely occurs through the stage of loading soluble N onto the nascent RNA strand as it exits the polymerase during RNA replication. Interfering with the oligomerization of P may represent a general strategy to interfere with NNS RNA virus replication.

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Yellow Fever Virus Reemergence and Spread in Southeast Brazil, 2016–2019

Journal of Virology ◽

10.1128/jvi.01623-19 ◽

2019 ◽

Vol 94 (1) ◽

Cited By ~ 8

Author(s):

Marta Giovanetti ◽

Marcos Cesar Lima de Mendonça ◽

Vagner Fonseca ◽

Maria Angélica Mares-Guia ◽

Allison Fabri ◽

...

Keyword(s):

Genetic Diversity ◽

Yellow Fever ◽

Rio De Janeiro ◽

Nonhuman Primates ◽

Yellow Fever Virus ◽

Genomic Data ◽

Fever Virus ◽

Southeastern Brazil ◽

Espirito Santo ◽

Southeastern Region

ABSTRACT 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.

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Retinoic Acid Inducible Gene I and Protein Kinase R, but Not Stress Granules, Mediate the Proinflammatory Response to Yellow Fever Virus

Journal of Virology ◽

10.1128/jvi.00403-20 ◽

2020 ◽

Vol 94 (22) ◽

Author(s):

Guillaume Beauclair ◽

Felix Streicher ◽

Maxime Chazal ◽

Daniela Bruni ◽

Sarah Lesage ◽

...

Keyword(s):

Yellow Fever ◽

Yellow Fever Virus ◽

Rna Virus ◽

Stress Granules ◽

Cytokine Response ◽

Fever Virus ◽

Stress Granule ◽

Proinflammatory Response ◽

Granule Formation ◽

Proinflammatory Mediators

ABSTRACT Yellow fever virus (YFV) is an RNA virus primarily targeting the liver. Severe YF cases are responsible for hemorrhagic fever, plausibly precipitated by excessive proinflammatory cytokine response. Pathogen recognition receptors (PRRs), such as the cytoplasmic retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), and the viral RNA sensor protein kinase R (PKR), are known to initiate a proinflammatory response upon recognition of viral genomes. Here, we sought to reveal the main determinants responsible for the acute cytokine expression occurring in human hepatocytes following YFV infection. Using a RIG-I-defective human hepatoma cell line, we found that RIG-I largely contributes to cytokine secretion upon YFV infection. In infected RIG-I-proficient hepatoma cells, RIG-I was localized in stress granules. These granules are large aggregates of stalled translation preinitiation complexes known to concentrate RLRs and PKR and are so far recognized as hubs orchestrating RNA virus sensing. Stable knockdown of PKR in hepatoma cells revealed that PKR contributes to both stress granule formation and cytokine induction upon YFV infection. However, stress granule disruption did not affect the cytokine response to YFV infection, as assessed by small interfering RNA (siRNA)-knockdown-mediated inhibition of stress granule assembly. Finally, no viral RNA was detected in stress granules using a fluorescence in situ hybridization approach coupled with immunofluorescence. Our findings suggest that both RIG-I and PKR mediate proinflammatory cytokine induction in YFV-infected hepatocytes, in a stress granule-independent manner. Therefore, by showing the uncoupling of the cytokine response from the stress granule formation, our model challenges the current view in which stress granules are required for the mounting of the acute antiviral response. IMPORTANCE Yellow fever is a mosquito-borne acute hemorrhagic disease caused by yellow fever virus (YFV). The mechanisms responsible for its pathogenesis remain largely unknown, although increased inflammation has been linked to worsened outcome. YFV targets the liver, where it primarily infects hepatocytes. We found that two RNA-sensing proteins, RIG-I and PKR, participate in the induction of proinflammatory mediators in human hepatocytes infected with YFV. We show that YFV infection promotes the formation of cytoplasmic structures, termed stress granules, in a PKR- but not RIG-I-dependent manner. While stress granules were previously postulated to be essential platforms for immune activation, we found that they are not required for the production of proinflammatory mediators upon YFV infection. Collectively, our work uncovered molecular events triggered by the replication of YFV, which could prove instrumental in clarifying the pathogenesis of the disease, with possible repercussions for disease management.

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