We describe an Ebolavirus minigenome-based system that is suitable for high-throughput screening of compounds able to impair Ebolavirus virus replication and/or transcription. The assay is robust (Z' factor,>0.6) and can be carried out in low-biosafety containment. Results from a pilot screen of 960 compounds are presented.

In this study, we used an RNA polymerase I (Pol I) transcription system for development of a reverse genetics protocol to produce hepatitis C virus (HCV), which is an uncapped positive-strand RNA virus. Transfection with a plasmid harboring HCV JFH-1 full-length cDNA flanked by a Pol I-promoter and Pol I-terminator yielded an unspliced RNA with no additional sequences at either end, resulting in efficient RNA replication within the cytoplasm and subsequent production of infectious virions. Using this technology, we developed a simple replicon trans-packaging system, in which transient transfection of two plasmids enables examination of viral genome replication and virion assembly as two separate steps. In addition, we established a stable cell line that constitutively produces HCV with a low mutation frequency of the viral genome. The effects of inhibitors of N-linked glycosylation on HCV production were evaluated using this cell line, and the results suggest that certain step(s), such as virion assembly, intracellular trafficking, and secretion, are potentially up- and down-regulated according to modifications of HCV envelope protein glycans. This Pol I-based HCV expression system will be beneficial for a high-throughput antiviral screening and vaccine discovery programs.

The advent of reverse genetics technology has revolutionized the study of RNA viruses, making it possible to manipulate their genomes and evaluate the effects of these changes on their biology and pathogenesis. The fundamental insights gleaned from reverse genetics-based studies over the last several years provide a new momentum for the development of designed therapies for the control and prevention of these viral pathogens. This review summarizes the successes and stumbling blocks in the development of reverse genetics technologies for Rift Valley fever virus and their application to the further dissection of its pathogenesis and the design of new therapeutics and safe and effective vaccines.

We establish a plasmid-driven minigenome system for Newcastle disease virus (NDV) V4 strain. Unlike the previously reported T7 polymerase based rescue system for Mononegavirales, the developed strategy does not necessitate the introduction of exogenous T7 polymerase by helper virus or stably expressing cell lines. This was achieved by transfection of plasmid pCAGGS-T7. The open reading frame (ORF) of enhanced green-fluorescent protein (EGFP) gene was inserted into constructed minigenome system pBRT7-mini and has been successfully expressed. Further packaging experiments indicate that 3' end leader and 5' end trailer regions are important for replication, transcription and packaging.

Ebola virus (EBOV), a public health concern in Africa and a potential biological weapon, is classified as a biosafety level-4 agent because of its high mortality rate and the lack of approved vaccines and antivirals. Basic research into the mechanisms of EBOV pathogenicity and the development of effective countermeasures are restricted by the current biosafety classification of EBOVs. We therefore developed biologically contained EBOV that express a reporter gene instead of the VP30 gene, which encodes an essential transcription factor. A Vero cell line that stably expresses VP30 provides this essential protein in trans and biologically confines the virus to its complete replication cycle in this cell line. This complementation approach is highly efficient because biologically contained EBOVs lacking the VP30 gene grow to titers similar to those obtained with wild-type virus. Moreover, EBOVs lacking the VP30 gene are indistinguishable in their morphology from wild-type virus and are genetically stable, as determined by sequence analysis after seven serial passages in VP30-expressing Vero cells. We propose that this system provides a safe means to handle EBOV outside a biosafety level-4 facility and will stimulate critical studies on the EBOV life cycle as well as large-scale screening efforts for compounds with activity against this lethal virus.

The Filoviruses Marburg virus and Ebola virus are among the deadliest of human pathogens, causing fulminant hemorrhagic fevers typified by overmatched specific immune responses and profuse inflammatory responses. Keys to both vaccination and treatment may reside, first, in the understanding of immune dysfunctions that parallel Filoviral disease and, second, in devising ways to redirect and restore normal immune function as well as to mitigate inflammation. Here, we describe how Filoviral infections may subvert innate immune responses through perturbances of dendritic cells and neutrophils, with particular emphasis on the downstream effects on adaptive immunity and inflammation. We suggest that pivotal events may be subject to therapeutic intervention as Filoviruses encounter immune processes.

Being highly pathogenic for human and nonhuman primates and the subject of former weapon programmes makes Ebola virus one of the most feared pathogens worldwide today. Due to a lack of licensed pre- and postexposure intervention, the current response depends on rapid diagnostics, proper isolation procedures and supportive care of case patients. Consequently, the development of more specific countermeasures is of high priority for the preparedness of many nations. Over the past years, enhanced research efforts directed to better understand virus replication and pathogenesis have identified potential new targets for intervention strategies. The authors discuss the most promising therapeutic approaches for Ebola haemorrhagic fever as judged by their efficacy in animal models. The current development in this field encourages discussions on how to move some of the experimental approaches towards clinical application.

Infectious virus-like particle (iVLP) systems have recently been established for several negative-strand RNA viruses, including the highly pathogenic Zaire ebolavirus (ZEBOV), and allow study of the viral life cycle under biosafety level 2 conditions. However, current systems depend on the expression of viral helper nucleocapsid proteins in target cells, thus making it impossible to determine whether ribonucleoprotein complexes transferred by iVLPs are able to facilitate initial transcription, an indispensable step in natural infection. Here we describe a ZEBOV iVLP system which overcomes this limitation and show that VP24 is essential for the formation of a functional ribonucleoprotein complex.

The packaging of viral genomic RNA into nucleocapsids and subsequently into virions is not completely understood. Phosphoprotein (P) and nucleoprotein (NP) interactions link NP-RNA complexes with P-L (polymerase) complexes to form viral nucleocapsids. The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging of the nucleocapsid into the virion. A mammalian two-hybrid assay and confocal microscopy were used to demonstrate that Ebola virus VP35 and VP40 interact and colocalize in transfected cells. VP35 was packaged into budding virus-like particles (VLPs) as observed by protease protection assays. Moreover, VP40 and VP35 were sufficient for packaging an Ebola virus minignome RNA into VLPs. Results from immunoprecipitation-reverse transcriptase PCR experiments suggest that VP35 confers specificity of the nucleocapsid for viral genomic RNA by direct VP35-RNA interactions.

Background. Recent reports indicate the possibility of using small interfering RNAs (siRNAs) to treat filovirus infections; however, they also show that the effectiveness of this approach is highly dependent on target site selection. Therefore, we explored the application of minigenomes as screening tools to identify functional siRNA targets under biosafety level 2 conditions.Methods. siRNA candidates were screened using the minigenome system to identify those with potential antiviral activity, compared with controls with poor predicted function on the basis of design guidelines, or those that were noncomplementary to Zaire ebolavirus (ZEBOV). These findings were then validated in cell culture by use of a previously developed ZEBOV expressing green fluorescent protein (ZEBOV-GFP), which allowed siRNA function to be easily assessed via flow cytometry or focus formation.Results. The most promising siRNA based on minigenome screening, targeting the nucleoprotein (NP) mRNA (ZNP1), also reduced protein expression and decreased viral titers after infection with ZEBOV-GFP to an extent similar to that reported for an siRNA recently shown to be therapeutic in guinea pigs.Conclusions. Minigenome screening appears to be an effective and convenient method of evaluating the therapeutic potential of siRNA targets, and findings suggest that its use would increase success rates in later stages of siRNA testing.

To facilitate an understanding of the molecular aspects of the pathogenesis of Zaire ebolavirus (ZEBOV) infection, we generated 2 different recombinant viruses expressing enhanced green fluorescent protein (eGFP) from additional transcription units inserted at different positions in the virus genome. These viruses showed in vitro phenotypes similar to that of wild-type ZEBOV (wt-ZEBOV) and were stable over multiple passages. Infection with one of the viruses expressing eGFP produced only mild disease in rhesus macaques, demonstrating a marked attenuation in this animal model. However, in mice lacking signal transducer and activator of transcription 1, both viruses expressing eGFP caused lethal cases of disease that were moderately attenuated, compared with that caused by wt-ZEBOV. In mice, viral replication could be easily tracked by the detection of eGFP-positive cells in tissues, by use of flow cytometry. These findings demonstrate that the incorporation of a foreign gene will attenuate ZEBOV in vivo but that these viruses still have potential for in vitro and in vivo research applications.

Infectious virus-like particle (iVLP) systems have recently been established for several negative-strand RNA viruses, including the highly pathogenic Zaire ebolavirus (ZEBOV), and allow study of the viral life cycle under biosafety level 2 conditions. However, current systems depend on the expression of viral helper nucleocapsid proteins in target cells, thus making it impossible to determine whether ribonucleoprotein complexes transferred by iVLPs are able to facilitate initial transcription, an indispensable step in natural infection. Here we describe a ZEBOV iVLP system which overcomes this limitation and show that VP24 is essential for the formation of a functional ribonucleoprotein complex.

Using the infectious clone for Zaire ebolavirus, the functional specificity of viral proteins of the ribonucleoprotein complex in transcription/replication was investigated by substituting them with heterologous proteins derived from closely (Reston ebolavirus) and distantly related filoviruses (Marburgvirus). The data clearly demonstrated that transcription/replication are neither strictly species-specific nor genus-specific. Protein interactions between the nucleoprotein NP and the virion protein VP35 and the polymerase L and VP35 seemed to be the most critical steps. In contrast to previous data, viral proteins were able to target heterologous filovirus RNA. Together these results indicated that protein-protein interactions are more critical than protein-RNA interactions.

We have determined the entire genomic sequence of the Pennsylvania strain, which was isolated along with the Virginia strain during the emergence of Ebola virus Reston in 1989/90 in the United States. Thus, either the Pennsylvania or Virginia strain, neither of which had been previously molecularly characterized, can be considered as the prototype for Ebola virus Reston. Comparative analysis showed a high degree of homology to the concomitantly analyzed and recently published Philippine strain of EBOV Reston from 1996 (Ikegami et al., Arch. Virol., 146 (2001) 2021). In comparison to EBOV Zaire, strain Mayinga, conservation could be found within the open reading frames, the 3' leader and 5' trailer region and the transcriptional signals, whereas the non-coding and intergenic regions did not show any homology. This clearly supports that EBOV Reston is a distinct species within the genus Ebola-like virus but which seems to be similar to other members with respect to transcription and replication strategies. The sequence determination provides the basis for the development of a reverse genetics system for Ebola virus Reston, which is needed to study differences in pathogenicity among filoviruses.

Marburg virus belongs to the genus Marburgvirus in the family Filoviridae and causes a severe hemorrhagic fever, known as Marburg hemorrhagic fever (MHF), in both humans and nonhuman primates. Similar to the more widely known Ebola hemorrhagic fever, MHF is characterized by systemic viral replication, immunosuppression and abnormal inflammatory responses. These pathological features of the disease contribute to a number of systemic dysfunctions including hemorrhages, edema, coagulation abnormalities and, ultimately, multiorgan failure and shock, often resulting in death. A detailed understanding of the pathological processes that lead to this devastating disease remains elusive, a fact that contributes to the lack of licensed vaccines or effective therapeutics. This article will review the clinical aspects of MHF and discuss the pathogenesis and possible options for diagnosis, treatment and prevention.

For Ebola virus (EBOV), 4 different species are known: Zaire, Sudan, Côte d'Ivoire, and Reston ebolavirus. The newly discovered Bundibugyo ebolavirus has been proposed as a 5th species. So far, no cross-neutralization among EBOV species has been described, aggravating progress toward cross-species protective vaccines. With the use of recombinant vesicular stomatitis virus (rVSV)-based vaccines, guinea pigs could be protected against Zaire ebolavirus (ZEBOV) infection only when immunized with a vector expressing the homologous, but not a heterologous, EBOV glycoprotein (GP). However, infection of guinea pigs with nonadapted wild-type strains of the different species resulted in full protection of all animals against subsequent challenge with guinea pig-adapted ZEBOV, showing that cross-species protection is possible. New vectors were generated that contain EBOV viral protein 40 (VP40) or EBOV nucleoprotein (NP) as a second antigen expressed by the same rVSV vector that encodes the heterologous GP. After applying a 2-dose immunization approach, we observed an improved cross-protection rate, with 5 of 6 guinea pigs surviving the lethal ZEBOV challenge if vaccinated with rVSV-expressing SEBOV-GP and -VP40. Our data demonstrate that cross-protection between the EBOV species can be achieved, although EBOV-GP alone cannot induce the required immune response.

Human infections with Ebola virus (EBOV) result in a deadly viral disease known as Ebola hemorrhagic fever. Up to 90% of infected patients die, and there is no available treatment or vaccine. The sporadic human outbreaks are believed to result when EBOV "jumps" from an infected animal to a person and is subsequently transmitted between persons by direct contact with infected blood or body fluids. This study was undertaken to investigate the mechanism by which EBOV can persistently infect and then escape from model cell and animal reservoir systems. We report a model system in which infection of mouse and bat cell lines with EBOV leads to persistence, which can be broken with low levels of lipopolysaccharide or phorbol-12-myristate-13-acetate (PMA). This reactivation depends on the Ras/MAPK pathway through inhibition of RNA-dependent protein kinase and eukaryotic initiation factor 2alpha phosphorylation and occurs at the level of protein synthesis. EBOV also can be evoked from mice 7 days after infection by PMA treatment, indicating that a similar mechanism occurs in vivo. Our findings suggest that EBOV may persist in nature through subclinical infection of a reservoir species, such as bats, and that appropriate physiological stimulation may result in increased replication and transmission to new hosts. Identification of a presumptive mechanism responsible for EBOV emergence from its reservoir underscores the "hit-and-run" nature of the initiation of human and/or nonhuman primate EBOV outbreaks and may provide insight into possible countermeasures to interfere with transmission.

Since Ebola virus was first identified more than 30 years ago, tremendous progress has been made in understanding the molecular biology and pathogenesis of this virus. However, the means by which Ebola virus is maintained and transmitted in nature remains unclear despite dedicated efforts to answer these questions. Recent work has provided new evidence that fruit bats might have a role as a reservoir species, but it is not clear whether other species are also involved or how transmission to humans or apes takes place. Two opposing hypotheses for Ebola emergence have surfaced; one of long-term local persistence in a cryptic and infrequently contacted reservoir, versus another of a more recent introduction of the virus and directional spread through susceptible populations. Nevertheless, with the increasing frequency of human filovirus outbreaks and the tremendous impact of infection on the already threatened great ape populations, there is an urgent need to better understand the ecology of Ebola virus in nature.

Zaire ebolavirus (ZEBOV) causes severe hemorrhagic fever in humans and nonhuman primates, with fatality rates in humans of up to 90%. The molecular basis for the extreme virulence of ZEBOV remains elusive. While adult mice resist ZEBOV infection, the Mayinga strain of the virus has been adapted to cause lethal infection in these animals. To understand the pathogenesis underlying the extreme virulence of Ebola virus (EBOV), here we identified the mutations responsible for the acquisition of the high virulence of the adapted Mayinga strain in mice, by using reverse genetics. We found that mutations in viral protein 24 and in the nucleoprotein were primarily responsible for the acquisition of high virulence. Moreover, the role of these proteins in virulence correlated with their ability to evade type I interferon-stimulated antiviral responses. These findings suggest a critical role for overcoming the interferon-induced antiviral state in the pathogenicity of EBOV and offer new insights into the pathogenesis of EBOV infection.

To the Editor: Filoviruses cause highly lethal hemorrhagic fever in humans and nonhuman primates, except for Reston Ebolavirus (REBOV), which causes severe hemorrhagic fever in macaques (1,2). REBOV epizootics among cynomolgus macaques occurred in 1989, 1990, 1992, and 1996 (2) and among swine in 2008 (3). African fruit bats have been suggested to be natural reservoirs for Zaire Ebolavirus and Marburg virus (4-6). However, the natural reservoir of REBOV in the Philippines is unknown. Thus, we determined the prevalence of REBOV antibody-positive bats in the Philippines.

The glycoproteins (GP) of enveloped viruses facilitate entry into the host cell by interacting with specific cellular receptors. Despite extensive study, a cellular receptor for the deadly filoviruses Ebolavirus and Marburgvirus has yet to be identified and characterized. Here, we show that T-cell Ig and mucin domain 1 (TIM-1) binds to the receptor binding domain of the Zaire Ebola virus (EBOV) glycoprotein, and ectopic TIM-1 expression in poorly permissive cells enhances EBOV infection by 10- to 30-fold. Conversely, reduction of cell-surface expression of TIM-1 by RNAi decreased infection of highly permissive Vero cells. TIM-1 expression within the human body is broader than previously appreciated, with expression on mucosal epithelia from the trachea, cornea, and conjunctiva--tissues believed to be important during in vivo transmission of filoviruses. Recognition that TIM-1 serves as a receptor for filoviruses on these mucosal epithelial surfaces provides a mechanistic understanding of routes of entry into the human body via inhalation of aerosol particles or hand-to-eye contact. ARD5, a monoclonal antibody against the IgV domain of TIM-1, blocked EBOV binding and infection, suggesting that antibodies or small molecules directed against this cellular receptor may provide effective filovirus antivirals.

Ebola viruses (EBOV) are causative agents of lethal hemorrhagic fever in humans and non-human primates. Among the filoviruses characterized thus far, Reston Ebola virus (REBOV) is the only Ebola species that is non-pathogenic in humans despite the fact that REBOV can cause lethal disease in non-human primates. Previous studies also suggest that Reston EBOV is less effective at inhibiting host innate immune responses, compared with Zaire EBOV or Marburg virus. Virally encoded VP35 protein is critical for immune suppression, but an understanding of the relative contributions of VP35 proteins from REBOV and other filoviruses is currently lacking. In order to address this question, we characterized REBOV VP35 IFN inhibitory domain (IID) using structural, biochemical, and virological studies. These studies reveal differences in dsRNA binding and IFN inhibition between the two species. These observed differences are likely due to increased stability and loss of flexibility in REBOV VP35 IID as demonstrated by thermal-shift stability assays. Consistent with this finding, our 1.71 A crystal structure of the REBOV VP35 IID reveal that the structure is highly similar to ZEBOV VP35 IID with an overall backbone r.m.s.d. of 0.64 A, but contains an additional helical element at the linker between the two subdomains of VP35 IID. Mutations near the linker, including swapping sequences between REBOV and ZEBOV, reveal that the linker sequence has limited tolerance for variability. Together with the previously solved ligand free and dsRNA bound forms of ZEBOV VP35 IID structures, our current studies of REBOV VP35 IID reinforce the importance of VP35 in immune suppression. Functional differences observed between REBOV and ZEBOV VP35 proteins may contribute to observed differences in pathogenicity, but these are unlikely be the major determinant. However, the high similarity in structure and the low tolerance of sequence variability, coupled with the multiple critical roles played by EBOV VP35 proteins, highlight the viability of VP35 as a potential target for therapeutic development.

Typical reverse genetics systems for generating influenza viruses require the insertion of each genome segments by DNA ligation into vectors for genome synthesis and expression. Herein is described the construction and use of a novel pair of plasmid vectors for cloning all eight genome segments of influenza A virus by homologous recombination for influenza virus reverse genetics. Plasmids, pLLBA and pLLBG, were constructed to possess opposing RNA polymerase I and RNA polymerase II transcription units for generating influenza genomic and messenger RNAs, respectively. In addition these promoters flanked a recombination cassette which comprised the conserved 5'(13bp) and 3'(12bp) terminal promoters of influenza virus. These vectors differed due to the presence of an A or a G (plus sense) to correspond to differences at nucleotide position 4 among negative-sense influenza virus promoters. The cloning approach involved homologous recombination of each influenza gene segment and the appropriate linearized pLLBA or pLLBG vectors in E. coli. Direct cloning by recombination was simpler and faster than conventional restriction digestion and ligation methods. This new vector system was successfully used to clone and rescue various influenza viruses and thus has the potential to promote the rapid analysis and vaccine development of novel influenza strains.

We have established a human RNA polymerase I (pol I)-driven influenza virus reverse genetics (RG) system in the Madin-Darby canine kidney 33016-PF cell line, which is approved for influenza vaccine manufacture. RNA pol I polymerases are generally active only in cells of species closely related to the species of origin of the polymerases. Nevertheless, we show that a non-endogenous RNA pol I promoter drives efficient rescue of influenza A viruses in a canine cell line. Application of this system allows efficient generation of virus strains and presents an alternative approach for influenza vaccine production.

Hantaan virus (HTNV) is an Old World hantavirus associated with hemorrhagic fever with renal syndrome (HFRS). To visualize the localization of the L protein of HTNV strain 84FLi within cells, a fusion protein composed of enhanced green fluorescent protein and L protein, EGFP-L, was expressed in Vero cells. The 273 KDa expressed fusion protein of EGFP-L localized in the perinuclear region. We also described the development of a reverse genetics system for HTNV strain 84FLi. The RNA polymerase I (pol I)-mediated transcription system was used to generate artificial viral RNA genome segments (minigenomes), which contained the chloramphenicol acetyltransferase (CAT) reporter gene in antisense (virus RNA) or sense (virus-complementary RNA) orientation flanked by the noncoding regions of HTNV 84FLi L segment. CAT could be detected in cells after transfection, indicating the successful encapsidation, transcription and replication of the pol I-derived minigenomes. The passaged transfer of CAT demonstrates that recombinant virus containing packaged pol I-derived minigenomes has been produced. This system may be helpful in studying the gene function and pathogenesis of HTNV.

We establish a plasmid-driven minigenome system for Newcastle disease virus (NDV) V4 strain. Unlike the previously reported T7 polymerase based rescue system for Mononegavirales, the developed strategy does not necessitate the introduction of exogenous T7 polymerase by helper virus or stably expressing cell lines. This was achieved by transfection of plasmid pCAGGS-T7. The open reading frame (ORF) of enhanced green-fluorescent protein (EGFP) gene was inserted into constructed minigenome system pBRT7-mini and has been successfully expressed. Further packaging experiments indicate that 3' end leader and 5' end trailer regions are important for replication, transcription and packaging.

Recombinant vesicular stomatitis virus (VSV) vectors expressing homologous filoviral glycoproteins can completely protect rhesus monkeys against Marburg virus when administered after exposure and can partially protect macaques after challenge with Zaire ebolavirus. Here, we administered a VSV vector expressing the Sudan ebolavirus (SEBOV) glycoprotein to four rhesus macaques shortly after exposure to SEBOV. All four animals survived SEBOV challenge while a control animal that received a nonspecific vector developed fulminant SEBOV hemorrhagic fever and succumbed. This is the first demonstration of complete postexposure protection against an Ebola virus in nonhuman primates and provides further evidence that postexposure vaccination may have utility in treating exposures to filoviruses.

In this study we report the development and optimization of two minigenome rescue systems for Nipah virus, a member of the Paramyxoviridae family. One is mediated by the T7 RNA polymerase supplied either by a constitutively expressing cell line or by transfection of expression plasmids and is thus independent from infection with a helper virus. The other approach is based on RNA polymerase I-driven transcription, a unique approach for paramyxovirus reverse genetics technology. Minigenome rescue was evaluated by reporter gene activities of (i) the two different minigenome transcription systems,(ii) genomic versus antigenomic-oriented minigenomes,(iii) different ratios of the viral protein expression plasmids, and (iv) time course experiments. The high efficiency and reliability of the established systems allowed for downscaling to 96-well plates. This served as a basis for the development of a high-throughput screening system for potential antivirals that target replication and transcription of Nipah virus without the need of high bio-containment. Using this system we were able to identify two compounds that reduced minigenome activity.

The rescue of influenza viruses by reverse genetics has been described only for the influenza A and B viruses. Based on a similar approach we developped a reverse genetics system that allows the production of influenza C viruses entirely from cloned cDNA. The complete sequences of the 3' and 5' non coding regions of type C influenza virus C/Johannesburg/1/66 necessary for the cloning of the cDNA were determined for the seven genomic segments. Human embryonic kidney cells (293T) were transfected simultaneously with seven plasmids, that direct the synthesis of each of the seven viral RNA segments of the C/JHB/1/66 virus, under the control of the human RNA polymerase I promoter and with four plasmids encoding the viral nucleoprotein and the PB2, PB1, and P3 proteins of the viral polymerase complex. This strategy yielded between 10(3) and 10(4) plaque forming units of virus per ml of supernatant at 8-10 days post-transfection. Additional viruses with substitutions introduced in the HEF protein were successfully produced by this method, and their growth phenotype was evaluated. This efficient system, which does not require helper virus infection should be useful in viral mutagenesis studies and to generate expression vectors from type C influenza virus.