T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus

Rift Valley fever virus (RVFV) is responsible for large and recurrent outbreaks of acute febrile illness among humans and domesticated animals in Africa. It belongs to the family Bunyaviridae, genus Phlebovirus, and its negative-stranded RNA genome consists of three segments. Here, we report the establishment and characterization of two different systems to rescue the RVFV wild-type strain ZH548. The first system is based on the BHK-21 cell clone BSR-T7/5, which stably expresses T7 RNA polymerase (T7 pol). Rescue of wild-type RVFV was achieved with three T7 pol-driven cDNA plasmids representing the viral RNA segments in the antigenomic sense. The second system involves 293T cells transfected with three RNA pol I-driven plasmids for the viral segments and two RNA pol II-driven support plasmids to express the viral polymerase components L and N. It is known that the 5′ triphosphate group of T7 pol transcripts strongly activates the antiviral interferon system via the intracellular RNA receptor RIG-I. Nonetheless, both the T7 pol and the pol I/II system were of similar efficiency. This was even true for the rescue of a RVFV mutant lacking the interferon antagonist nonstructural proteins. Further experiments demonstrated that the unresponsiveness of BHK-21 and BSR-T7/5 cells to T7 pol transcripts is most probably due to a deficiency in the RIG-I pathway. Our reverse genetics systems now enable us to manipulate the genome of RVFV and study its virulence mechanisms. Moreover, the finding that BHK-derived cell lines have a compromised RIG-I pathway may explain their suitability for propagating and rescuing a wide variety of viruses.

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Generation and characterization of a recombinant Rift Valley fever virus expressing a V5 epitope-tagged RNA-dependent RNA polymerase

Journal of General Virology ◽

10.1099/vir.0.036749-0 ◽

2011 ◽

Vol 92 (12) ◽

pp. 2906-2913 ◽

Cited By ~ 13

Author(s):

Benjamin Brennan ◽

Ping Li ◽

Richard M. Elliott

Keyword(s):

Life Cycle ◽

Rna Polymerase ◽

Rift Valley ◽

Rift Valley Fever ◽

Rift Valley Fever Virus ◽

Fever Virus ◽

Rna Dependent Rna Polymerase ◽

Valley Fever ◽

L Protein ◽

Infected Cells

The viral RNA-dependent RNA polymerase (RdRp; L protein) of Rift Valley fever virus (RVFV; family Bunyaviridae) is a 238 kDa protein that is crucial for the life cycle of the virus, as it catalyses both transcription of viral mRNAs and replication of the tripartite genome. Despite its importance, little is known about the intracellular distribution of the polymerase or its other roles during infection, primarily because of lack of specific antibodies that recognize L protein. To begin to address these questions we investigated whether the RVFV (MP12 strain) polymerase could tolerate insertion of the V5 epitope, as has been previously demonstrated for the Bunyamwera virus L protein. Insertion of the 14 aa epitope into the polymerase sequence at aa 1852 resulted in a polymerase that retained functionality in a minigenome assay, and we were able to rescue recombinant viruses that expressed the modified L protein by reverse genetics. The L protein could be detected in infected cells by Western blotting with anti-V5 antibodies. Examination of recombinant virus-infected cells by immunofluorescence revealed a punctate perinuclear or cytoplasmic distribution of the polymerase that co-localized with the nucleocapsid protein. The generation of RVFV expressing a tagged RdRp will allow detailed examination of the role of the viral polymerase in the virus life cycle.

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Genetic Evidence for an Interferon-Antagonistic Function of Rift Valley Fever Virus Nonstructural Protein NSs

Journal of Virology ◽

10.1128/jvi.75.3.1371-1377.2001 ◽

2001 ◽

Vol 75 (3) ◽

pp. 1371-1377 ◽

Cited By ~ 246

Author(s):

Michèle Bouloy ◽

Christian Janzen ◽

Pierre Vialat ◽

Huot Khun ◽

Jovan Pavlovic ◽

...

Keyword(s):

Rift Valley ◽

Rift Valley Fever ◽

Nonstructural Protein ◽

Rift Valley Fever Virus ◽

Fever Virus ◽

Sub Saharan Africa ◽

Wild Type ◽

Valley Fever ◽

Viral Virulence ◽

Deficient Mice

ABSTRACT Rift Valley fever virus (RVFV), a phlebovirus of the family Bunyaviridae, is a major public health threat in Egypt and sub-Saharan Africa. The viral and host cellular factors that contribute to RVFV virulence and pathogenicity are still poorly understood. All pathogenic RVFV strains direct the synthesis of a nonstructural phosphoprotein (NSs) that is encoded by the smallest (S) segment of the tripartite genome and has an undefined accessory function. In this report, we show that MP12 and clone 13, two attenuated RVFV strains with mutations in the NSs gene, were highly virulent in IFNAR−/− mice lacking the alpha/beta interferon (IFN-α/β) receptor but remained attenuated in IFN-γ receptor-deficient mice. Both attenuated strains proved to be excellent inducers of early IFN-α/β production. In contrast, the virulent strain ZH548 failed to induce detectable amounts of IFN-α/β and replicated extensively in both IFN-competent and IFN-deficient mice. Clone 13 has a defective NSs gene with a large in-frame deletion. This defect in the NSs gene results in expression of a truncated protein which is rapidly degraded. To investigate whether the presence of the wild-type NSs gene correlated with inhibition of IFN-α/β production, we infected susceptible IFNAR−/− mice with S gene reassortant viruses. When the S segment of ZH548 was replaced by that of clone 13, the resulting reassortants became strong IFN inducers. When the defective S segment of clone 13 was exchanged with the wild-type S segment of ZH548, the reassortant virus lost the capacity to stimulate IFN-α/β production. These results demonstrate that the ability of RVFV to inhibit IFN-α/β production correlates with viral virulence and suggest that the accessory protein NSs is an IFN antagonist.

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Characterization of Wild-Type and Alternate Transcription Termination Signals in the Rift Valley Fever Virus Genome

Journal of Virology ◽

10.1128/jvi.05322-11 ◽

2011 ◽

Vol 85 (23) ◽

pp. 12134-12145 ◽

Cited By ~ 22

Author(s):

E. Lara ◽

A. Billecocq ◽

P. Leger ◽

M. Bouloy

Keyword(s):

Rift Valley ◽

Rift Valley Fever ◽

Rift Valley Fever Virus ◽

Transcription Termination ◽

Virus Genome ◽

Fever Virus ◽

Wild Type ◽

Valley Fever

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Rift Valley Fever Virus MP-12 Vaccine Is Fully Attenuated by a Combination of Partial Attenuations in the S, M, and L Segments

Journal of Virology ◽

10.1128/jvi.00135-15 ◽

2015 ◽

Vol 89 (14) ◽

pp. 7262-7276 ◽

Cited By ~ 28

Author(s):

Tetsuro Ikegami ◽

Terence E. Hill ◽

Jennifer K. Smith ◽

Lihong Zhang ◽

Terry L. Juelich ◽

...

Keyword(s):

United States ◽

Rift Valley ◽

Rift Valley Fever ◽

Rift Valley Fever Virus ◽

The United States ◽

Fever Virus ◽

Pathogenic Strain ◽

Wild Type ◽

Valley Fever ◽

Content Type

ABSTRACTRift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and characterized by a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. RVF is caused by Rift Valley fever virus (RVFV; familyBunyaviridae, genusPhlebovirus), which has a tripartite negative-stranded RNA genome (consisting of the S, M, and L segments). Further spread of RVF into countries where the disease is not endemic may affect the economy and public health, and vaccination is an effective approach to prevent the spread of RVFV. A live-attenuated MP-12 vaccine is one of the best-characterized RVF vaccines for safety and efficacy and is currently conditionally licensed for use for veterinary purposes in the United States. Meanwhile, as of 2015, no other RVF vaccine has been conditionally or fully licensed for use in the United States. The MP-12 strain is derived from wild-type pathogenic strain ZH548, and its genome encodes 23 mutations in the three genome segments. However, the mechanism of MP-12 attenuation remains unknown. We characterized the attenuation of wild-type pathogenic strain ZH501 carrying a mutation(s) of the MP-12 S, M, or L segment in a mouse model. Our results indicated that MP-12 is attenuated by the mutations in the S, M, and L segments, while the mutations in the M and L segments confer stronger attenuation than those in the S segment. We identified a combination of 3 amino acid changes, Y259H (Gn), R1182G (Gc), and R1029K (L), that was sufficient to attenuate ZH501. However, strain MP-12 with reversion mutations at those 3 sites was still highly attenuated. Our results indicate that MP-12 attenuation is supported by a combination of multiple partial attenuation mutations and a single reversion mutation is less likely to cause a reversion to virulence of the MP-12 vaccine.IMPORTANCERift Valley fever (RVF) is a mosquito-transmitted viral disease that is endemic to Africa and that has the potential to spread into other countries. Vaccination is considered an effective way to prevent the disease, and the only available veterinary RVF vaccine in the United States is a live-attenuated MP-12 vaccine, which is conditionally licensed. Strain MP-12 is different from its parental pathogenic RVFV strain, strain ZH548, because of the presence of 23 mutations. This study determined the role of individual mutations in the attenuation of the MP-12 strain. We found that full attenuation of MP-12 occurs by a combination of multiple mutations. Our findings indicate that a single reversion mutation will less likely cause a major reversion to virulence of the MP-12 vaccine.

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Safety and Immunogenicity of Four-Segmented Rift Valley Fever Virus in the Common Marmoset

10.21203/rs.3.rs-1075403/v1 ◽

2021 ◽

Author(s):

Paul Wichgers Schreur ◽

Petra Mooij ◽

Gerrit Koopman ◽

Babs Verstrepen ◽

Zahra Fagrouch ◽

...

Keyword(s):

Rift Valley ◽

Rift Valley Fever ◽

Rift Valley Fever Virus ◽

Common Marmoset ◽

Fever Virus ◽

Wild Type ◽

Valley Fever ◽

Veterinary Vaccine ◽

The Common ◽

Human Vaccine

Abstract Rift Valley fever virus (RVFV) is an emerging mosquito-borne bunyavirus that is highly pathogenic to wild- and domesticated ruminants, camelids and humans. While animals are exclusively infected via mosquito bites, humans can also be infected via contact with tissues or blood released during the slaughtering of RVFV-infected animals. No human vaccine is available and currently commercialized veterinary vaccines do not optimally combine efficacy with safety. We previously reported the development of two novel live-attenuated RVF vaccines, created by splitting the M genome segment and deleting the major virulence determinant NSs. The vaccine candidates, referred to as the veterinary vaccine vRVFV-4s and the human vaccine hRVFV-4s, were shown to induce protective immunity in multiple species after a single vaccination. Anticipating on accidental exposure of humans to the veterinary vaccine, and to evaluate the safety of the hRVFV-4s candidate vaccine for humans, the safety of each vaccine was evaluated in the most susceptible nonhuman primate model, the common marmoset (Callithrix jacchus). Marmosets were inoculated with high doses of each vaccine and were monitored for clinical signs as well as for vaccine virus dissemination, shedding and spreading to the environment. To accurately assess the attenuation of both vaccine viruses, separate groups of marmosets were inoculated with the parent wild-type RVFV strains. Both wild-type strains induced high viremia and disseminated to primary target organs, associated with mild- to severe morbidity, while both vaccines were well tolerated with absence of dissemination and shedding, while inducing potent neutralizing antibody responses. The results of the studies support the unprecedented safety profile of both vaccines for animals and humans.

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