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Expression of GFP in tissues sectioned from HeV-GFP challenged ferrets. Ferrets were challenged with 5,000 TCID50 of HeV-GFP and succumbed to disease on days 7 and 8. At necropsy, kidney (a,b,c) and lung (d,e,f) samples were collected, fixed with paraformaldehye, sectioned and imaged by confocal microscopy. To assess sensitive of GFP expression (green; a and d) to traditional staining techniques, HeV nucleocapsid protein (red; b and e) was co-stained using rabbit anti- HeV N sera detected with anti-rabbit conjugated Alexafluor 568. Composite images (c and f) show both GFP and HeV N labelling. Scale bars: c =25 μm, f = 40 μm.
Source publication
Background
Hendra virus (HeV) is an Australian bat-borne zoonotic paramyxovirus that repeatedly spills-over to horses causing fatal disease. Human cases have all been associated with close contact with infected horses.
Methods
A full-length antigenome clone of HeV was assembled, a reporter gene (GFP or luciferase) inserted between the P and M gene...
Citations
... Further studies will explore to which extent the W assembly into fibrils participate to W function during Henipavirus infection in cellula and in vivo. The quick formation of W fibrils may benefit to the virus to fight against the cell innate immune response in light of the short timescale of viral infections, the duration of which is limited to few hours in vitro (43) and few days in vivo after experimental infection (44). Viral amyloids represent a new promising antiviral target considering their emerging roles in virus replication (45). ...
The Hendra and Nipah viruses (HeV and NiV) are zoonotic biosafety level-4 pathogens within the Paramyxoviridae family. We previously showed that their W proteins form amyloid-like fibrils in vitro . Here, we demonstrate that W also forms fibrils in cellula and that cysteine residues are crucial in dictating the ability of W proteins to fibrillate. The cysteine oxidation state acts as a switch to generate either amorphous aggregates or flexible fibrils. Ectopic expression of W HeV induces an oxidative stress and W HeV fibrils were observed in the nuclei of different cell lines, with fibrillation being impaired by cysteine substitutions. Finally, nuclear fibrils are associated with an impairment of the NF-κB pathway in W HeV transfected cells. This work provides experimental evidence for the ability of Henipavirus W proteins to fibrillate in transfected cells and the first clues on their functional impact.
Significance Statement
Nipah and Hendra viruses are severe pathogens infecting humans and livestock, classified among the 8 highest priorities for research by the WHO. The W protein, along with the V protein, is a virulence factor responsible for antiviral response inhibition and we demonstrate here that its fibrillation into amyloid-like fibrils occurs in the nucleus of transfected cells, with their formation being dependent of the redox state of the W cysteine residues. The sole transfection of W provokes the production of reactive oxygen species, creating a suitable environment for the fibrils to form. Finally, we show that W fibrils enhance the repression of the antiviral response, thus pointing to W fibrillation as a new promising antiviral target.
... and efficacy of candidate countermeasures. A number of additional viruses have also been evaluated in the ferret model with varying degrees of success, including Middle East respiratory syndrome coronavirus, 31 rabies virus, [32][33][34] Hendra virus, 35,36 Nipah virus, 37 mumps virus, 38-40 simian virus 5, 41 and canine 42 and human parainfluenza viruses (reviewed in Enkirch and von Messing). 43 More recently, the ferret has become increasingly popular as a model for filoviruses, particularly for evaluating pathogenesis and transmission as well as for efficacy testing of candidate countermeasures. ...
The domestic ferret (Mustela putorius furo) has long been a popular animal model for evaluating viral pathogenesis and transmission as well as the efficacy of candidate countermeasures. Without question, the ferret has been most widely implemented for modeling respiratory viruses, particularly influenza viruses; however, in recent years, it has gained attention as a novel animal model for characterizing filovirus infections. Although ferrets appear resistant to infection and disease caused by Marburg and Ravn viruses, they are highly susceptible to lethal disease caused by Ebola, Sudan, Bundibugyo, and Reston viruses. Notably, unlike the immunocompetent rodent models of filovirus infection, ferrets are susceptible to lethal disease caused by wild-type viruses, and they recapitulate many aspects of human filovirus disease, including systemic virus replication, coagulation abnormalities, and a dysregulated immune response. Along with the stringency with which they reproduce Ebola disease, their relatively small size and availability make ferrets an attractive choice for countermeasure evaluation and pathogenesis modeling. Indeed, they are so far the only small animal model available for Bundibugyo virus. Nevertheless, ferrets do have their limitations, including the lack of commercially available reagents to dissect host responses and their unproven predictive value in therapeutic evaluation. Although the use of the ferret model in ebolavirus research has been consistent over the last few years, its widespread use and utility remains to be fully proven. This review provides a comprehensive overview of the ferret models of filovirus infection and perspective on their ongoing use in pathogenesis modeling and countermeasure evaluation.
... Direct detection and visualization of infected cells by fluorescent protein expression significantly reduces the technical aspects of both IHC and flow cytometry. The advantages to this approach have been highlighted in key studies using fluorescent recombinant reporter viruses in animal models of disease to investigate cellular targets of infection, tissue tropism, and viral dissemination for multiple pathogens including measles [12], henipaviruses [13], and influenza viruses [14]. ...
Crimean-Congo hemorrhagic fever virus (CCHFV, order Bunyavirales, family Nairoviridae, genus Orthonairovirus) is the tick-borne etiological agent of Crimean-Congo hemorrhagic fever (CCHF) in humans. Animals are generally susceptible to CCHFV infection but refractory to disease. Small animal models are limited to interferon-deficient mice, that develop acute fatal disease following infection. Here, using a ZsGreen1- (ZsG) expressing reporter virus (CCHFV/ZsG), we examine tissue tropism and dissemination of virus in interferon-α/β receptor knock-out (Ifnar-/-) mice. We demonstrate that CCHFV/ZsG retains in vivo pathogenicity comparable to wild-type virus. Interestingly, despite high levels of viral RNA in all organs assessed, 2 distribution patterns of infection were observed by both fluorescence and immunohistochemistry (IHC), corresponding to the permissiveness of organ tissues. To further investigate viral dissemination and to temporally define cellular targets of CCHFV in vivo, mice were serially euthanized at different stages of disease. Flow cytometry was used to characterize CCHFV-associated alterations in hematopoietic cell populations and to classify infected cells in the blood, lymph node, spleen, and liver. ZsG signal indicated that mononuclear phagocytic cells in the lymphatic tissues were early targets of infection; in late-stage infection, overall, the highest levels of signal were detected in the liver, and ZsG was found in both antigen-presenting and lymphocyte cell populations.
... The genomes of NiV-M and NiV-B have the same organization, encoding a nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), glycoprotein (G), and large protein (L) 37 ; and despite the pathogenic differences between NiV-M and NiV-B 38 , the two genotypes share an overall nucleotide homology of 91.8% 11 . Reverse genetics systems have been developed for HeV 39,42 , Cedar virus, a non-pathogenic henipavirus 40 , and NiV-M [41][42][43] , which has enabled insightful functional studies of henipavirus pathogenesis [42][43][44][45][46] ; however, despite NiV-B being the agent responsible for every outbreak since 1998/99, with the possible exception of an outbreak in the Philippines in 2014 23 , a reverse genetics system for NiV-B has yet to be reported. The development of recombinant NiV-M encoding a marker gene such as EGFP or luciferase has enabled high-throughput drug screening 47,48 . ...
... The development of recombinant NiV-M encoding a marker gene such as EGFP or luciferase has enabled high-throughput drug screening 47,48 . Strategies to develop recombinant henipaviruses have evolved over the last 10 years: the initial recombinant henipavirus (NiV-M) expressing a foreign reporter gene was generated with the marker gene inserted between the N and P genes 41 ; however since foreign gene insertion can interfere with the polar transcription gradient of paramyxoviruses 49 subsequent recombinant henipaviruses have been constructed with the marker gene placed between the P and M genes 39,40 or at the 5′ end of the M gene to generate a self-cleaved fusion protein 42,47 . Further, the initial recombinant NiV depended on recombinant vaccinia virus that expressed the bacteriophage T7 polymerase (T7pol) for rescue 41 , whereas successive recombinant henipaviruses have employed co-transfection with a T7pol expression construct 39,42 or rescue in a cell line that stably expresses T7pol 43 . ...
... Strategies to develop recombinant henipaviruses have evolved over the last 10 years: the initial recombinant henipavirus (NiV-M) expressing a foreign reporter gene was generated with the marker gene inserted between the N and P genes 41 ; however since foreign gene insertion can interfere with the polar transcription gradient of paramyxoviruses 49 subsequent recombinant henipaviruses have been constructed with the marker gene placed between the P and M genes 39,40 or at the 5′ end of the M gene to generate a self-cleaved fusion protein 42,47 . Further, the initial recombinant NiV depended on recombinant vaccinia virus that expressed the bacteriophage T7 polymerase (T7pol) for rescue 41 , whereas successive recombinant henipaviruses have employed co-transfection with a T7pol expression construct 39,42 or rescue in a cell line that stably expresses T7pol 43 . Additional technical refinements have improved rescue efficiency such as optimized self-cleaving hammerhead ribozyme sequences 42 and optimized P2A ribosomal skipping/cleavage sequence 50 , required to process transcribed RNA to match vRNA sequences. ...
Nipah virus (NiV) has emerged as a highly lethal zoonotic paramyxovirus that is capable of causing a febrile encephalitis and/or respiratory disease in humans for which no vaccines or licensed treatments are currently available. There are two genetically and geographically distinct lineages of NiV: NiV-Malaysia (NiV-M), the strain that caused the initial outbreak in Malaysia, and NiV-Bangladesh (NiV-B), the strain that has been implicated in subsequent outbreaks in India and Bangladesh. NiV-B appears to be both more lethal and have a greater propensity for person-to-person transmission than NiV-M. Here we describe the generation and characterization of stable RNA polymerase II-driven infectious cDNA clones of NiV-M and NiV-B. In vitro, reverse genetics-derived NiV-M and NiV-B were indistinguishable from a wildtype isolate of NiV-M, and both viruses were pathogenic in the Syrian hamster model of NiV infection. We also describe recombinant NiV-M and NiV-B with enhanced green fluorescent protein (EGFP) inserted between the G and L genes that enable rapid and sensitive detection of NiV infection in vitro. This panel of molecular clones will enable studies to investigate the virologic determinants of henipavirus pathogenesis, including the pathogenic differences between NiV-M and NiV-B, and the high-throughput screening of candidate therapeutics.
... In other words, a foreign antigen should be expressed at a proper, but not high, level. To date, the P/M intergenic region in paramyxoviruses has been widely used for insertion of foreign antigens into cDNA clones (Banyard et al., 2010;Cantarella et al., 2009;Marsh et al., 2013;Wang et al., 2012;Xu et al., 2017), and so has this region in this study (Fig. 1). ...
... To develop a platform to understand pathogenesis of henipaviruses, we used a reverse genetics approach to rescue replication-competent, recombinant CedPV (rCedPV). Reverse genetic systems have been utilized for the generation of recombinant infectious and replication-competent negative sense RNA viruses with specific mutations and insertions [58,59], particularly NiV and HeV [60][61][62][63][64]. Introduction of reporter genes, such as green fluorescent protein (GFP) or luciferase, provides for an ability to monitor virus replication and spread in real time and/or to perform high-throughput screening [63]. ...
... To develop a platform to understand pathogenesis of henipaviruses, we used a reverse genetics approach to rescue replication-competent, recombinant CedPV (rCedPV). Reverse genetic systems have been utilized for the generation of recombinant infectious and replication-competent negative sense RNA viruses with specific mutations and insertions [58,59], particularly NiV and HeV [60][61][62][63][64]. Introduction of reporter genes, such as green fluorescent protein (GFP) or luciferase, provides for an ability to monitor virus replication and spread in real time and/or to perform high-throughput screening [63]. In this study, we describe the rescue of two rCedPV variants, one recombinant wild-type CedPV (rCedPV-wt) and one of which expresses GFP from an additional open reading frame (rCedPV-GFP). ...
... These fragments were sequentially cloned into an expression plasmid, pOLTV5 [65], between the T7 RNA promoter and hepatitis delta virus (HDV) ribozyme. The pOLTV5 vector was similarly used for cloning and expression of recombinant HeV [63]. At the time of the rCedPV cDNA clone design, the nucleotide at position 7 in the CedPV reference genome was a cytosine, which was later revised to an adenine. ...
Background:
Hendra virus and Nipah virus are zoonotic viruses that have caused severe to fatal disease in livestock and human populations. The isolation of Cedar virus, a non-pathogenic virus species in the genus Henipavirus, closely-related to the highly pathogenic Hendra virus and Nipah virus offers an opportunity to investigate differences in pathogenesis and receptor tropism among these viruses.
Methods:
We constructed full-length cDNA clones of Cedar virus from synthetic oligonucleotides and rescued two replication-competent, recombinant Cedar virus variants: a recombinant wild-type Cedar virus and a recombinant Cedar virus that expresses a green fluorescent protein from an open reading frame inserted between the phosphoprotein and matrix genes. Replication kinetics of both viruses and stimulation of the interferon pathway were characterized in vitro. Cellular tropism for ephrin-B type ligands was qualitatively investigated by microscopy and quantitatively by a split-luciferase fusion assay.
Results:
Successful rescue of recombinant Cedar virus expressing a green fluorescent protein did not significantly affect virus replication compared to the recombinant wild-type Cedar virus. We demonstrated that recombinant Cedar virus stimulated the interferon pathway and utilized the established Hendra virus and Nipah virus receptor, ephrin-B2, but not ephrin-B3 to mediate virus entry. We further characterized virus-mediated membrane fusion kinetics of Cedar virus with the known henipavirus receptors ephrin-B2 and ephrin-B3.
Conclusions:
The recombinant Cedar virus platform may be utilized to characterize the determinants of pathogenesis across the henipaviruses, investigate their receptor tropisms, and identify novel pan-henipavirus antivirals. Moreover, these experiments can be conducted safely under BSL-2 conditions.
... After optimizing the rSOSV rescue system, we designed another recombinant SOSV genome capable of expressing a fluorescent reporter protein in infected cells. Previous approaches to develop recombinant paramyxoviruses expressing reporter genes have included the reporter as either a separate open reading frame (ORF), or incorporated it within a parental ORF [18][19][20][21][22]. We employed the latter approach, and generated a SOSV genome in which the ZsG ORF was inserted upstream of the M ORF, fused to the viral protein via the self-cleaving P2A motif taken from porcine teschovirus 1 [23]. ...
... A recent study focusing on only 10% of bat species in relatively small geographic areas discovered evidence of 66 novel paramyxoviruses [28], so undiscovered pathogens with the capacity to threaten human health may yet be present in bat populations worldwide. With reverse genetics systems already developed for the highly pathogenic paramyxoviruses NiV and HeV, the establishment of SOSV reverse genetics based on similar design concepts ensures that any investigations into future outbreaks can be quickly initiated [20,21,29]. Furthermore, a common feature of paramyxoviruses appears to be that expression of reporter genes does not negatively affect the viral phenotype either in vitro or in vivo, increasing the usefulness of these systems [21,22,30,31]. ...
... With reverse genetics systems already developed for the highly pathogenic paramyxoviruses NiV and HeV, the establishment of SOSV reverse genetics based on similar design concepts ensures that any investigations into future outbreaks can be quickly initiated [20,21,29]. Furthermore, a common feature of paramyxoviruses appears to be that expression of reporter genes does not negatively affect the viral phenotype either in vitro or in vivo, increasing the usefulness of these systems [21,22,30,31]. The minigenome screening methods described here were successfully used to identify several compounds that inhibit SOSV replication. ...
Sosuga virus (SOSV) is a recently discovered zoonotic paramyxovirus isolated from a single human case in 2012; it has been ecologically and epidemiologically associated with transmission by the Egyptian rousette bat (Rousettus aegyptiacus). Bats have long been recognized as sources of novel zoonotic pathogens, including highly lethal paramyxoviruses like Nipah virus (NiV) and Hendra virus (HeV). The ability of SOSV to cause severe human disease supports the need for studies on SOSV pathogenesis to better understand the potential impact of this virus and to identify effective treatments. Here we describe a reverse genetics system for SOSV comprising a minigenome-based assay and a replication-competent infectious recombinant reporter SOSV that expresses the fluorescent protein ZsGreen1 in infected cells. First, we used the minigenome assay to rapidly screen for compounds inhibiting SOSV replication at biosafety level 2 (BSL-2). The antiviral activity of candidate compounds was then tested against authentic viral replication using the reporter SOSV at BSL-3. We identified several compounds with anti-SOSV activity, several of which also inhibit NiV and HeV. Alongside its utility in screening for potential SOSV therapeutics, the reverse genetics system described here is a powerful tool for analyzing mechanisms of SOSV pathogenesis, which will facilitate our understanding of how to combat the potential public health threats posed by emerging bat-borne paramyxoviruses.
... The large number of HeV incidents in Australia from 2011 to 2013 prompted researchers at our laboratory to establish the capability to perform genome-wide RNAi screens at BSL-4. Central to this work was the development of a recombinant HeV expressing the renilla luciferase construct, which allowed for high throughput and rapid measurement of virus infection (Marsh et al. 2013). This recombinant virus was shown to be lethal in the ferret model of henipavirus disease and exhibited a pathogenesis profile comparable to the wild-type virus. ...
Hendra and Nipah viruses (family Paramyxoviridae, genus Henipavirus) are zoonotic RNA viruses that cause lethal disease in humans and are designated as Biosafety Level 4 (BSL4) agents. Moreover, henipaviruses belong to the same group of viruses that cause disease more commonly in humans such as measles, mumps and respiratory syncytial virus. Due to the relatively recent emergence of the henipaviruses and the practical constraints of performing functional genomics studies at high levels of containment, our understanding of the henipavirus infection cycle is incomplete. In this chapter we describe recent loss-of-function (i.e. RNAi) functional genomics screens that shed light on the henipavirus–host interface at a genome-wide level. Further to this, we cross-reference RNAi results with studies probing host proteins targeted by henipavirus proteins, such as nuclear proteins and immune modulators. These functional genomics studies join a growing body of evidence demonstrating that nuclear and nucleolar host proteins play a crucial role in henipavirus infection. Furthermore these studies will underpin future efforts to define the role of nucleolar host–virus interactions in infection and disease.
... *, P Ͻ 0.05; **, P Ͻ 0.01. previously been used to drive successful rescue of HeV and Crimean Zaire hemorrhagic fever virus (CCHFV); in the latter case, codon optimization of the viral RdRp L gene was critical for efficient rescue (19,20). A plasmid-based method for delivering T7 polymerase is advantageous because it theoretically allows for the use of any transfectable cell type. ...
The notoriously low efficiency of Paramyxoviridae reverse genetics systems has posed a limiting barrier to the study of viruses in this family. Previous approaches to reverse genetics have utilized a wide variety of techniques to overcome the technical hurdles. Although robustness (i.e., the number of attempts that result in successful rescue) has been improved in some systems with the use of stable cell lines, the efficiency of rescue (i.e., the proportion of transfected cells that yield at least one successful rescue event) has remained low. We have substantially increased rescue efficiency for representative viruses from all five major Paramyxoviridae genera (from ~1 in 10⁶-10⁷ to ~1 in 10²-10³ transfected cells) by the addition of a self-cleaving hammerhead ribozyme (Hh-Rbz) sequence immediately preceding the start of the recombinant viral antigenome and the use of a codon-optimized T7 polymerase (T7opt) gene to drive paramyxovirus rescue. Here, we report a strategy for robust, reliable, and high-efficiency rescue of paramyxovirus reverse genetics systems, featuring several major improvements: (i) a vaccinia virus-free method, (ii) freedom to use any transfectable cell type for viral rescue, (iii) a single-step transfection protocol, and (iv) use of the optimal T7 promoter sequence for high transcription levels from the antigenomic plasmid without incorporation of nontemplated G residues. The robustness of our T7opt-HhRbz system also allows for greater latitude in the ratios of transfected accessory plasmids used that result in successful rescue. Thus, our system may facilitate the rescue and interrogation of the increasing number of emerging paramyxoviruses.
... The lysine residue, identified as a site for ubiquitination, is indicated by an asterisk.henipaviruses, the M proteins from HeV, NiV, CedV, MojV and KV (GH-74a virus) fused to GFP were expressed in transiently transfected HeLa cells, which support the full life cycle of NiV and HeV[5,11,35]. Contrarily, MeV-M protein is a predominantly cytoplasmic protein, with a small percentage of protein detected in the nucleus in transfected or MeV-infected cells[36][37][38]. Kidney cells (PaKi) derived from the bat host (Pteropus alecto), which support virus replication, were also examined to determine host-specific differences. ...
Viruses of the genus Henipavirus of the family Paramyxoviridae are zoonotic pathogens, which have emerged in South East Asia, Australia and Africa. Nipah virus (NiV) and Hendra virus (HeV) are highly virulent pathogens transmitted from bats to animals and humans, whilst the henipavirus Cedar virus (CedV) seems to be non-pathogenic in infection studies. The full replication cycle of the Paramyxoviridae occurs in the host cell's cytoplasm where viral assembly is orchestrated by the matrix (M) protein. Unexpectedly, the NiV-M protein traffics through the nucleus as an essential step to engage the plasma membrane in preparation for viral budding/release. Comparative studies were performed to assess whether M protein nuclear localization is a common feature of the henipaviruses including the recently sequenced (although not yet isolated) Ghanaian bat henipavirus (Kumasi virus, GH-M74a virus, KV) and Mojiang virus (MojV). Live-cell confocal microscopy revealed that nuclear translocation of GFP-fused M protein is conserved between henipaviruses in both human and bat-derived cell lines. However, the efficiency of M protein nuclear localization and virus-like particle budding competency varied. Additionally, CedV-, KV- and MojV-M proteins were mutated in a bipartite nuclear localization signal indicating that a key lysine residue is essential for nuclear import, export and for the induction of budding events as previously reported for NiV-M. The results of this study suggest that the M proteins of henipaviruses may utilize a similar nucleocytoplasmic trafficking pathway as an essential step during viral replication in both humans and bats.
