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.

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.

The prototypic arenavirus lymphocytic choriomeningitis virus has been a primary workhorse of viral immunologists for almost a century, and it has served as an important model for studying basic principles of arenavirus molecular biology. Its negative-stranded bisegmented RNA genome has, however, posed a major obstacle to attempts at manipulating the infectious virus by reverse genetic techniques. Here, we report the recovery of infectious lymphocytic choriomeningitis virus (the immunosuppressive strain clone 13) entirely from cDNA. Intracellular transcription of the short and the long viral genome segment from polymerase (pol) I-driven vectors and coexpression of the minimal viral-transacting factors NP and L from pol II-driven plasmids resulted in the efficient formation of infectious virus with genetic tags in both genome segments. The cDNA-derived viruses behaved identically to wild-type virus in both cell culture and infected mice. Importantly, they caused a chronic infection and suppressed the adaptive immune response to an unrelated third-party virus. This technology provides an important basis for investigating viral determinants of persistent infection and immunosuppression. In addition, our findings demonstrate that pol I/II-based vector systems may represent an efficient alternative strategy for the recovery of cytoplasmic negative-strand RNA viruses from cDNA.

Here we report recovery of infectious Marburg virus (MARV) from a full-length cDNA clone. Compared to the wild-type virus, recombinant MARV showed no difference in terms of morphology of virus particles, intracellular distribution in infected cells, and growth kinetics. The nucleocapsid protein VP30 of MARV and Ebola virus (EBOV) contains a Zn-binding motif which is important for the function of VP30 as a transcriptional activator in EBOV, whereas its role for MARV is unclear. It has been reported previously that MARV VP30 is able to support transcription in an EBOV-specific minigenome system. When the Zn-binding motif was destroyed, MARV VP30 was shown to be inactive in the EBOV system. While it was not possible to rescue recombinant MARV when the VP30 plasmid was omitted from transfection, MARV VP30 with a destroyed Zn-binding motif and EBOV VP30 were able to mediate virus recovery. In contrast, rescue of recombinant EBOV was not supported by EBOV VP30 containing a mutated Zn-binding domain.

Ebola virus (EBOV), an emerging pathogen, is the causative agent of a rapidly progressive hemorrhagic fever with high mortality rates. There are currently no approved vaccines or treatments available for Ebola hemorrhagic fever. Standard plaque assays are currently the only reliable techniques for enumerating the virus. Effective drug-discovery screening as well as target identification and validation require simple and more rapid detection methods. This report describes the development of a rapid ELISA that measures virus release with high sensitivity. This assay detects both Ebola virus and EBOV-like particles (VLPs) directly from cell-culture supernatants with the VP40 matrix protein serving as antigen. Using this assay, the contribution of the EBOV nucleocapsid (NC) proteins in VLP release was determined. These findings indicate that a combination of NC proteins together with the envelope components is optimal for VLP formation and release, a finding that is important for vaccination with Ebola VLPs. Furthermore, this assay can be used in surrogate models in non-biocontainment environment, facilitating both basic research on the mechanism of EBOV assembly and budding as well as drug-discovery research.

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.

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.

Ebola virus (EBOV) causes severe haemorrhagic fever leading to up to 90% lethality. Increasingly frequent outbreaks and the placement of EBOV in the category A list of potential biothreat agents have boosted interest in this virus. Furthermore, development of new technologies (e.g. reverse genetics systems) and extensive studies on Ebola haemorrhagic fever (EHF) in animal models have substantially expanded the knowledge on the pathogenic mechanisms that underlie this disease. Two major factors in EBOV pathogenesis are the impairment of the immune response and vascular dysfunction. Here, we attempt to summarize the current knowledge on EBOV pathogenesis focusing on these two factors and on recent progress in the development of vaccines and potential therapeutics.

Viral hemorrhagic fever (VHF) is an infectious syndrome in humans often associated with high fatality rates. For most VHFs there are no specific and effective therapies or vaccines available and, in general, there is a lack of knowledge regarding the biology and pathogenesis of the causative agents. Therefore, a more detailed understanding of the molecular basis of VHF pathogenesis, including the identification of viral virulence determinants and host interactions and responses, will be important to enhance our ability to control VHF infections. The recently developed "reverse genetics systems" for several VHF causing viruses have allowed the generation of infectious viruses from cloned cDNA and thus, the generation of virus mutants. Here we review the existing reverse genetics systems for VHF causing viruses and discuss their use in studying viral replication, pathogenesis, and the development of antivirals and vaccines.

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.

Reverse genetics system is an excellent platform to research the construction and function of viruses. Genome modification, such as gene recombination, mosaicism, and mutation may interfere with replication, assembly and release of viruses. An efficient, convenient and economical method of virus rescue is undoubtedly required for elevating the efficiency of rescuing crippled virus. In this study, we developed a method to rescue infectious bursal disease virus (IBDV) using RNA polymerase II. The genome of IBDV Gt strain, flanked by hammerhead ribozyme and hepatitis delta ribozyme sequences, were cloned downstream of the cytomegalovirus enhancer and the beta chicken actin promoter of the vector pCAGGS. Through direct transfection in various cell lines, IBDV could be rescued efficiently. The RNA polymerase II-based reverse genetics system is efficient, stable, convenient, and fit to various cells. The system not only provides the basis of the gene function research of IBDV, but is also useful for reverse genetics research of other birnaviridae viruses.

The currently available reverse-genetics systems for Influenza A virus are all based on transcription of genomic RNA by RNA polymerase I, but the species specificity of this polymerase is a disadvantage. A reverse-genetics vector containing a T7 RNA polymerase promoter, hepatitis delta virus ribozyme sequence and T7 RNA polymerase terminator sequence has been developed. To achieve optimal expression in minigenome assays, it was determined that viral RNA should be inserted in this vector in the negative-sense orientation with two additional G residues downstream of the T7 RNA polymerase promoter. It was also shown that expression of the minigenome was more efficient when a T7 RNA polymerase with a nuclear-localization signal was used. By using this reverse-genetics system, recombinant influenza virus A/PR/8/34 was produced more efficiently than by using a similar polymerase I-based reverse-genetics system. Furthermore, influenza virus A/NL/219/03 could be rescued from 293T, MDCK and QT6 cells. Thus, a reverse-genetics system for the rescue of Influenza A virus has been developed, which will be useful for fundamental research and vaccine seed strain production in a variety of cell lines.