Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome, causes a mosquito-borne disease that is endemic in sub-Saharan African countries and that also causes large epidemics among humans and livestock. Furthermore, it is a bioterrorist threat and poses a risk for introduction to other areas. In spite of its danger, neither veterinary nor human vaccines are available. We established a T7 RNA polymerase-driven reverse genetics system to rescue infectious clones of RVFV MP-12 strain entirely from cDNA, the first for any phlebovirus. Expression of viral structural proteins from the protein expression plasmids was not required for virus rescue, whereas NSs protein expression abolished virus rescue. Mutants of MP-12 partially or completely lacking the NSs open reading frame were viable. These NSs deletion mutants replicated efficiently in Vero and 293 cells, but not in MRC-5 cells. In the latter cell line, accumulation of beta interferon mRNA occurred after infection by these NSs deletion mutants, but not after infection by MP-12. The NSs deletion mutants formed larger plaques than MP-12 did in Vero E6 cells and failed to shut off host protein synthesis in Vero cells. An MP-12 mutant carrying a luciferase gene in place of the NSs gene replicated as efficiently as MP-12 did, produced enzymatically active luciferase during replication, and stably retained the luciferase gene after 10 virus passages, representing the first demonstration of foreign gene expression in any bunyavirus. This reverse genetics system can be used to study the molecular virology of RVFV, assess current vaccine candidates, produce new vaccines, and incorporate marker genes into animal vaccines.     

The dominant-negative inhibition of double-stranded RNA-dependent protein kinase PKR increases the efficacy of Rift Valley fever virus MP-12 vaccine

Rift Valley fever virus (RVFV), belonging to the genus Phlebovirus, family Bunyaviridae, is endemic to sub-Saharan Africa and causes a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. MP-12 is the only RVFV strain excluded from the select-agent rule and handled at a biosafety level 2 (BSL2) laboratory. MP-12 encodes a functional major virulence factor, the NSs protein, which contributes to its residual virulence in pregnant ewes. We found that 100% of mice subcutaneously vaccinated with recombinant MP-12 (rMP12)-murine PKRN167 (mPKRN167), which encodes a dominant-negative form of mouse double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in place of NSs, were protected from wild-type (wt) RVFV challenge, while 72% of mice vaccinated with MP-12 were protected after challenge. rMP12-mPKRN167 induced alpha interferon (IFN-α) in sera, accumulated RVFV antigens in dendritic cells at the local draining lymph nodes, and developed high levels of neutralizing antibodies, while parental MP-12 induced neither IFN-α nor viral-antigen accumulation at the draining lymph node yet induced a high level of neutralizing antibodies. The present study suggests that the expression of a dominant-negative PKR increases the immunogenicity and efficacy of live-attenuated RVFV vaccine, which will lead to rational design of safe and highly immunogenic RVFV vaccines for livestock and humans.     

Structural studies of Hantaan virus

Hantaan virus is the prototypic member of the Hantavirus genus within the family Bunyaviridae and is a causative agent of the potentially fatal hemorrhagic fever with renal syndrome. The Bunyaviridae are a family of negative-sense RNA viruses with three-part segmented genomes. Virions are enveloped and decorated with spikes derived from a pair of glycoproteins (Gn and Gc). Here, we present cryo-electron tomography and single-particle cryo-electron microscopy studies of Hantaan virus virions. We have determined the structure of the tetrameric Gn-Gc spike complex to a resolution of 2.5 nm and show that spikes are ordered in lattices on the virion surface. Large cytoplasmic extensions associated with each Gn-Gc spike also form a lattice on the inner surface of the viral membrane. Rod-shaped ribonucleoprotein complexes are arranged into nearly parallel pairs and triplets within virions. Our results differ from the T=12 icosahedral organization found for some bunyaviruses. However, a comparison of our results with the previous tomographic studies of the nonpathogenic Tula hantavirus indicates a common structural organization for hantaviruses.     

Rift Valley fever virus

Rift Valley fever is considered to be one of the most important viral zoonoses in Africa. In 2000, the Rift valley fever virus spread to the Arabian Peninsula and caused two simultaneous outbreaks in Yemen and Saudi Arabia. It is transmitted to ruminants and to humans by mosquitoes. The viral agent is an arbovirus, which belongs to the Phlebovirus genus in the Bunyaviridae family. This family of viruses comprises more than 300 members grouped into five genera: Orthobunyavirus, Phlebovirus, Hantavirus, Nairovirus, and Tospovirus. Several members of the Bunyaviridae family are responsible for fatal hemorrhagic fevers: Rift Valley fever virus (Phlebovirus), Crimean-Congo hemorrhagic fever virus (Nairovirus), Hantaan, Sin Nombre and related viruses (Hantavirus), and recently Garissa, now identified as Ngari virus (Orthobunyavirus). Here are reviewed recent advances in Rift Valley fever virus, its epidemiology, molecular biology and focus on recent data on the interactions between viral and cellular proteins, which help to understand the molecular mechanisms utilized by the virus to circumvent the host cellular response.     

Development of a Novel,Single-Cycle Replicable Rift Valley Fever Vaccine

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is an arbovirus that causes severe disease in humans and livestock in sub-Saharan African countries. Although the MP-12 strain of RVFV is a live attenuated vaccine candidate, neuroinvasiveness and neurovirulence of MP-12 in mice may be a concern when vaccinating certain individuals, especially those that are immunocompromised. We have developed a novel, single-cycle replicable MP-12 (scMP-12), which carries an L RNA, M RNA mutant encoding a mutant envelope protein lacking an endoplasmic reticulum retrieval signal and defective for membrane fusion function, and S RNA encoding N protein and green fluorescent protein. The scMP-12 underwent efficient amplification, then formed plaques and retained the introduced mutation after serial passages in a cell line stably expressing viral envelope proteins. However, inoculation of the scMP-12 into naïve cells resulted in a single round of viral replication, and production of low levels of noninfectious virus-like particles. Intracranial inoculation of scMP-12 into suckling mice did not cause clinical signs or death, a finding which demonstrated that the scMP-12 lacked neurovirulence. Mice immunized with a single dose of scMP-12 produced neutralizing antibodies, whose titers were higher than in mice immunized with replicon particles carrying L RNA and S RNA encoding N protein and green fluorescent protein. Moreover, 90% of the scMP-12-immunized mice were protected from wild-type RVFV challenge by efficiently suppressing viremia and replication of the challenge virus in the liver and the spleen. These data demonstrated that scMP-12 is a safe and immunogenic RVFV vaccine candidate.     

Creation of a nonspreading Rift Valley fever virus

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic bunyavirus of the genus Phlebovirus and a serious human and veterinary pathogen. RVFV contains a three-segmented RNA genome, which is comprised of the large (L), medium (M), and small (S) segments. The proteins that are essential for genome replication are encoded by the L and S segments, whereas the structural glycoproteins are encoded by the M segment. We have produced BHK replicon cell lines (BHK-Rep) that maintain replicating L and S genome segments. Transfection of BHK-Rep cells with a plasmid encoding the structural glycoproteins results in the efficient production of RVFV replicon particles (RRPs). To facilitate monitoring of infection, the NSs gene was replaced with an enhanced green fluorescent protein gene. RRPs are infectious for both mammalian and insect cells but are incapable of autonomous spreading, rendering their application outside biosafety containment completely safe. We demonstrate that a single intramuscular vaccination with RRPs protects mice from a lethal dose of RVFV and show that RRPs can be used for rapid virus neutralization tests that do not require biocontainment facilities. The methods reported here will greatly facilitate vaccine and drug development as well as fundamental studies on RVFV biology. Moreover, it may be possible to develop similar systems for other members of the bunyavirus family as well.     

Rift Valley fever virus 78kDa envelope protein attenuates virus replication in macrophage-derived cell lines and viral virulence in mice

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus with a wide host range including ruminants and humans. RVFV outbreaks have had devastating effects on public health and the livestock industry in African countries. However, there is no approved RVFV vaccine for human use in non-endemic countries and no FDA-approved antiviral drug for RVFV treatment. The RVFV 78kDa protein (P78), which is a membrane glycoprotein, plays a role in virus dissemination in the mosquito host, but its biological role in mammalian hosts remains unknown. We generated an attenuated RVFV MP-12 strain-derived P78-High virus and a virulent ZH501 strain-derived ZH501-P78-High virus, both of which expressed a higher level of P78 and carried higher levels of P78 in the virion compared to their parental viruses. We also generated another MP-12-derived mutant virus (P78-KO virus) that does not express P78. MP-12 and P78-KO virus replicated to similar levels in fibroblast cell lines and Huh7 cells, while P78-High virus replicated better than MP-12 in Vero E6 cells, fibroblast cell lines, and Huh7 cells. Notably, P78-High virus and P78-KO virus replicated less efficiently and more efficiently, respectively, than MP-12 in macrophage cell lines. ZH501-P78-High virus also replicated poorly in macrophage cell lines. Our data further suggest that inefficient binding of P78-High virus to the cells led to inefficient virus internalization, low virus infectivity and reduced virus replication in a macrophage cell line. P78-High virus and P78-KO virus showed lower and higher virulence than MP-12, respectively, in young mice. ZH501-P78-High virus also exhibited lower virulence than ZH501 in mice. These data suggest that high levels of P78 expression attenuate RVFV virulence by preventing efficient virus replication in macrophages. Genetic alteration leading to increased P78 expression may serve as a novel strategy for the attenuation of RVFV virulence and generation of safe RVFV vaccines.     

The capsid of small papova viruses contains 72 pentameric capsomeres: direct evidence from cryo-electron-microscopy of simian virus 40.

The three-dimensional structure of the simian virus 40 capsid is remarkably similar to the structure of the polyoma empty capsid. This similarity is apparent despite striking differences in the methods used to determine the two structures: image analysis of electron micrographs of frozen-hydrated samples (SV40 virions) and an unconventional x-ray crystallographic analysis (polyoma empty capsids). Both methods have clearly resolved the 72 prominent capsomere units which comprise the T = 7d icosahedral capsid surface lattice. The 12 pentavalent and 60 hexavalent capsomeres consist of pentameric substructures. A pentameric morphology for hexavalent capsomeres clearly shows that the conserved bonding specificity expected from the quasi-equivalence theory is not present in either SV40 or polyoma capsids. Determination of the SV40 structure from cryo-electron microscopy supports the correctness of the polyoma structure solved crystallographically and establishes a strong complementarity of the two techniques. Similarity between the SV40 virion and the empty polyoma capsid indicates that the capsid is not detectably altered by the loss of the nucleohistone core. The unexpected pentameric substructure of the hexavalent capsomeres and the arrangement of the 72 pentamers in the SV40 and polyoma capsid lattices may be characteristic features of all members of the papova virus family, including the papilloma viruses such as human wart and rabbit papilloma.     

Rift Valley Fever Virus Strain MP-12 Enters Mammalian Host Cells via Caveola-Mediated Endocytosis

Rift Valley fever virus (RVFV) is a zoonotic pathogen capable of causing serious morbidity and mortality in both humans and livestock. The lack of efficient countermeasure strategies, the potential for dispersion into new regions, and the pathogenesis in humans and livestock make RVFV a serious public health concern. The receptors, cellular factors, and entry pathways used by RVFV and other members of the family Bunyaviridae remain largely uncharacterized. Here we provide evidence that RVFV strain MP-12 uses dynamin-dependent caveola-mediated endocytosis for cell entry. Caveolae are lipid raft domains composed of caveolin (the main structural component), cholesterol, and sphingolipids. Caveola-mediated endocytosis is responsible for the uptake of a wide variety of host ligands, as well as bacteria, bacterial toxins, and a number of viruses. To determine the cellular entry mechanism of RVFV, we used small-molecule inhibitors, RNA interference (RNAi), and dominant negative (DN) protein expression to inhibit the major mammalian cell endocytic pathways. Inhibitors and RNAi specific for macropinocytosis and clathrin-mediated endocytosis had no effect on RVFV infection. In contrast, inhibitors of caveola-mediated endocytosis, and RNAi targeted to caveolin-1 and dynamin, drastically reduced RVFV infection in multiple cell lines. Expression of DN caveolin-1 also reduced RVFV infection significantly, while expression of DN EPS15, a protein required for the assembly of clathrin-coated pits, and DN PAK-1, an obligate mediator of macropinocytosis, had no significant impact on RVFV infection. These results together suggest that the primary mechanism of RVFV MP-12 uptake is dynamin-dependent, caveolin-1-mediated endocytosis.