• Paramyxoviridae
• Paramyxovirus
• Reverse genetics
• Virus rescue

• Animals
• Humans
• Paramyxoviridae/genetics
• Reverse Genetics/methods
• Animals, Wild
• Public Health
• RNA, Viral
• Paramyxoviridae Infections/epidemiology
• Phylogeny

• Cedar virus
• Henipavirus
• Reverse genetics
• Virus plaque assay
• Virus rescue
• Virus titration

• Henipavirus
• Reverse Genetics
• Orthopoxvirus
• Antiviral Agents

• U19 AI142764 NIAID NIH HHS
• U01 AI077995 NIAID NIH HHS
• R01 AI054715 NIAID NIH HHS

• CDLR-based mutagenesis
• RNA structure
• bioinformatics
• foot-and-mouth disease virus (FMDV)
• viral replication

• Foot-and-Mouth Disease Virus/enzymology
• Foot-and-Mouth Disease Virus/genetics
• Genome, Viral
• Mutagenesis
• Open Reading Frames
• RNA, Viral/chemistry
• RNA, Viral/genetics
• RNA-Dependent RNA Polymerase/genetics
• RNA-Dependent RNA Polymerase/metabolism

• BBS/E/I/00007035 Biotechnology and Biological Sciences Research Council
• BBS/E/I/00007036 Biotechnology and Biological Sciences Research Council
• BBS/E/I/00007037 Biotechnology and Biological Sciences Research Council
• BB/K003801/1 Biotechnology and Biological Sciences Research Council
• Department for Environment, Food and Rural Affairs, UK Government (Defra)
• UKRI | Biotechnology and Biological Sciences Research Council (BBSRC)

Generation of recombinant negative-stranded RNA viruses (NSVs) from plasmids involves in vivo reconstitution of biologically active nucleocapsids and faces a unique antisense problem where the negative-sense viral genomic RNAs can hybridize to viral messenger RNAs. To overcome this problem, a positive-sense RNA approach has been devised through expression of viral antigenomic (ag)RNA and core proteins for assembly of antigenomic nucleocapsids. Although this detour strategy works for many NSVs, the process is still inefficient. Using Sonchus yellow net rhabdovirus (SYNV) as a model; here, we develop a negative-sense genomic RNA-based approach that increased rescue efficiency by two orders of magnitude compared to the conventional agRNA approach. The system relied on suppression of double-stranded RNA induced antiviral responses by co-expression of plant viruses-encoded RNA silencing suppressors or animal viruses-encoded double-stranded RNA antagonists. With the improved approach, we were able to recover a highly attenuated SYNV mutant with a deletion in the matrix protein gene which otherwise could not be rescued via the agRNA approach. Reverse genetics analyses of the generated mutant virus provided insights into SYNV virion assembly and morphogenesis. This approach may potentially be applicable to other NSVs of plants or animals.

• Sonchus yellow net virus
• antigenome
• genome
• infectious clone
• matrix protein
• negative-stranded RNA virus
• nucleocapsid
• plant rhabdovirus
• recovery
• viral suppressor of RNA silencing

• Gene Expression Regulation, Viral
• Genes, Reporter
• Genetic Engineering
• Genome, Viral
• Plant Diseases/genetics
• Plant Diseases/virology
• Plant Viruses/genetics
• RNA Interference
• RNA, Viral
• Rhabdoviridae/genetics
• Sonchus/virology
• Transcription, Genetic

• HRSV
• Pathogenesis
• clinical isolates
• cotton rat
• respiratory system
• subtype A/B

• Animals
• Cell Line
• Coinfection/virology
• Female
• Genotype
• Green Fluorescent Proteins/genetics
• Green Fluorescent Proteins/metabolism
• Humans
• Luminescent Proteins/genetics
• Luminescent Proteins/metabolism
• Lung/virology
• Respiratory Mucosa/virology
• Respiratory Syncytial Virus Infections/virology
• Respiratory Syncytial Virus, Human/classification
• Respiratory Syncytial Virus, Human/genetics
• Respiratory Syncytial Virus, Human/isolation & purification
• Respiratory Syncytial Virus, Human/physiology
• Respiratory System/virology
• Respiratory Tract Infections/virology
• Reverse Genetics
• Sigmodontinae
• Red Fluorescent Protein

• G0801001 Medical Research Council

Many traditional vaccines have proven to be incapable of controlling newly emerging infectious diseases. They have also achieved limited success in the fight against a variety of human cancers. Thus, innovative vaccine strategies are highly needed to overcome the global burden of these diseases. Advances in molecular biology and reverse genetics have completely restructured the concept of vaccinology, leading to the emergence of state-of-the-art technologies for vaccine design, development and delivery. Among these modern vaccine technologies are the recombinant viral vectored vaccines, which are known for their incredible specificity in antigen delivery as well as the induction of robust immune responses in the vaccinated hosts. Although a number of viruses have been used as vaccine vectors, genetically engineered Newcastle disease virus (NDV) possesses some useful attributes that make it a preferable candidate for vectoring vaccine antigens. Here, we review the molecular biology of NDV and discuss the reverse genetics approaches used to engineer the virus into an efficient vaccine vector. We then discuss the prospects of the engineered virus as an efficient vehicle of vaccines against cancer and several infectious diseases of man and animals.

• Newcastle disease virus
• cancer
• infectious diseases
• reverse genetics
• vaccines

• Animals
• Cancer Vaccines/genetics
• Cancer Vaccines/immunology
• Genetic Engineering
• Genetic Vectors/genetics
• Genome, Viral
• Humans
• Newcastle disease virus/genetics
• Newcastle disease virus/immunology
• Oncolytic Viruses/genetics
• Oncolytic Viruses/immunology
• Ruminants
• Vaccines, Synthetic/genetics
• Vaccines, Synthetic/immunology
• Vaccinology/methods
• Virulence

• Ebola virus
• Marburg virus
• filoviruses
• genome replication and transcription
• nonsegmented negative sense RNA viruses

• Filoviridae/genetics
• Genome, Viral/genetics
• Regulatory Elements, Transcriptional/genetics
• Regulatory Elements, Transcriptional/physiology
• Virus Replication/genetics
• Virus Replication/physiology

• R01 AI133486 NIAID NIH HHS
• R21 AI137793 NIAID NIH HHS
• UC7 AI095321 NIAID NIH HHS

Ectopic protein with proper steric structure was efficiently loaded onto the envelope of the F gene-defective BC-PIV vector derived from human parainfluenza virus type 2 (hPIV2) by a reverse genetics method of recombinant virus production. Further, ectopic antigenic peptide was successfully loaded either outside, inside, or at both sides of the envelope of the vector. The BC-PIV vector harboring the Ebola virus GP gene was able to elicit neutralizing antibodies in mice. In addition, BC-PIV with antigenic epitopes of both melanoma gp100 and WT1 tumor antigen induced a CD8+ T-cell-mediated response in tumor-transplanted syngeneic mice. Considering the low pathogenicity and recurrent infections of parental hPIV2, BC-PIV can be used as a versatile vector with high safety for recombinant vaccine development, addressing unmet medical needs.

• Barley yellow striate mosaic virus
• cereals
• genomic studies
• plant rhabdovirus
• planthopper

• Animals
• Base Sequence
• DNA, Complementary/genetics
• Edible Grain/genetics
• Edible Grain/virology
• Gene Editing
• Genetic Vectors/metabolism
• Genome, Plant
• Genomics
• Glucuronidase/metabolism
• Hemiptera/virology
• Hordeum/ultrastructure
• Hordeum/virology
• Plant Leaves/virology
• Plant Viruses/physiology
• Plant Viruses/ultrastructure
• RNA, Guide, CRISPR-Cas Systems/metabolism
• Rhabdoviridae/physiology
• Rhabdoviridae/ultrastructure
• Nicotiana/ultrastructure
• Nicotiana/virology

Newcastle disease (ND) is one of the most devastating diseases that considerably cripple the global poultry industry. Because of its enormous socioeconomic importance and potential to rapidly spread to naïve birds in the vicinity, ND is included among the list of avian diseases that must be notified to the OIE immediately upon recognition. Currently, virus isolation followed by its serological or molecular identification is regarded as the gold standard method of ND diagnosis. However, this method is generally slow and requires specialised laboratory with biosafety containment facilities, making it of little relevance under epidemic situations where rapid diagnosis is seriously needed. Thus, molecular based diagnostics have evolved to overcome some of these difficulties, but the extensive genetic diversity of the virus ensures that isolates with mutations at the primer/probe binding sites escape detection using these assays. This diagnostic dilemma leads to the emergence of cutting-edge technologies such as next-generation sequencing (NGS) which have so far proven to be promising in terms of rapid, sensitive, and accurate recognition of virulent Newcastle disease virus (NDV) isolates even in mixed infections. As regards disease control strategies, conventional ND vaccines have stood the test of time by demonstrating track record of protective efficacy in the last 60 years. However, these vaccines are unable to block the replication and shedding of most of the currently circulating phylogenetically divergent virulent NDV isolates. Hence, rationally designed vaccines targeting the prevailing genotypes, the so-called genotype-matched vaccines, are highly needed to overcome these vaccination related challenges. Among the recently evolving technologies for the development of genotype-matched vaccines, reverse genetics-based live attenuated vaccines obviously appeared to be the most promising candidates. In this review, a comprehensive description of the current and emerging trends in the detection, identification, and control of ND in poultry are provided. The strengths and weaknesses of each of those techniques are also emphasised.

Cellular kinases are crucial for the transcription/replication of many negative-strand RNA viruses and might serve as targets for antiviral therapy. In this study, a library comprising 80 kinase inhibitors was screened for antiviral activity against vesicular stomatitis virus (VSV), a prototype member of the family Rhabdoviridae. 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125), an inhibitor of eukaryotic elongation factor 2 (eEF2) kinase, significantly inhibited entry of single-cycle VSV encoding a luciferase reporter. Treatment of virus particles had only minimal effect on virus entry, indicating that the compound primarily acts on the host cell rather than on the virus. Accordingly, resistant mutant viruses were not detected when the virus was passaged in the presence of the drug. Unexpectedly, NH125 led to enhanced, rather than reduced, phosphorylation of eEF2, however, it did not significantly affect cellular protein synthesis. In contrast, NH125 revealed lysosomotropic features and showed structural similarity with N-dodecylimidazole, a known lysosomotropic agent. Related alkylated imidazolium compounds also exhibited antiviral activity, which was critically dependent on the length of the alkyl group. Apart from VSV, NH125 inhibited infection by VSV pseudotypes containing the envelope glycoproteins of viruses that are known to enter cells in a pH-dependent manner, i.e. avian influenza virus (H5N1), Ebola virus, and Lassa virus. In conclusion, we identified an alkylated imidazolium compound which inhibited entry of several viruses not because of the previously postulated inhibition of eEF2 kinase but most likely because of its lysosomotropic properties.

• alkylated imidazolium
• enveloped virus
• eukaryotic elongation factor 2 kinase
• ionic liquid
• kinase inhibitor
• lysosomotropic agent
• membrane fusion
• virus entry

Reverse genetics is key technology for producing wild-type and genetically modified viruses. The ISA (Infectious Subgenomic Amplicons) method is a recent versatile and user-friendly reverse genetics method to rescue RNA viruses. The main constraint of its canonic protocol was the requirement to produce (e.g., by DNA synthesis or fusion PCR) 5' and 3' modified genomic fragments encompassing the human cytomegalovirus promoter (pCMV) and the hepatitis delta virus ribozyme/simian virus 40 polyadenylation signal (HDR/SV40pA), respectively. Here, we propose the ultimately simplified "Haiku" designs in which terminal pCMV and HDR/SV40pA sequences are provided as additional separate DNA amplicons. This improved procedure was successfully applied to the rescue of a wide range of viruses belonging to genera Flavivirus, Alphavirus and Enterovirus in mosquito or mammalian cells using only standard PCR amplification techniques and starting from a variety of original materials including viral RNAs extracted from cell supernatant media or animal samples. We also demonstrate that, in specific experimental conditions, the presence of the HDR/SV40pA is not necessary to rescue the targeted viruses. These ultimately simplified "Haiku" designs provide an even more simple, rapid, versatile and cost-effective tool to rescue RNA viruses since only generation of overlapping amplicons encompassing the entire viral genome is now required to generate infectious virus. This new approach may completely modify our capacity to obtain infectious RNA viruses.

• 35S promoter
• Hammerhead ribozyme
• Infectious clone
• Minireplicon
• Reverse genetics
• Rhabdovirus
• Sonchus yellow net virus
• Virus rescue

• Caulimovirus/genetics
• Gene Expression
• Plasmids
• Promoter Regions, Genetic
• RNA, Catalytic/metabolism
• RNA, Viral/genetics
• RNA, Viral/isolation & purification
• Rhabdoviridae/genetics
• Rhabdoviridae/isolation & purification
• Sonchus
• Nicotiana/virology
• Transcription, Genetic
• Virology/methods

• National Natural Science Foundation of China
• National Basic Research Program of China

The ability to manipulate the genome of paramyxoviruses and evaluate the effects of these changes at the phenotypic level is a powerful tool for the investigation of specific aspects of the viral life cycle and viral pathogenesis. However, reverse genetics systems for paramyxoviruses are notoriously inefficient, when successful. The ability to efficiently and robustly rescue paramyxovirus reverse genetics systems can be used to answer basic questions about the biology of paramyxoviruses, as well as to facilitate the considerable translational efforts being devoted to developing live attenuated paramyxovirus vaccine vectors.

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.

IMPORTANCE The ability to manipulate the genome of paramyxoviruses and evaluate the effects of these changes at the phenotypic level is a powerful tool for the investigation of specific aspects of the viral life cycle and viral pathogenesis. However, reverse genetics systems for paramyxoviruses are notoriously inefficient, when successful. The ability to efficiently and robustly rescue paramyxovirus reverse genetics systems can be used to answer basic questions about the biology of paramyxoviruses, as well as to facilitate the considerable translational efforts being devoted to developing live attenuated paramyxovirus vaccine vectors.

• paramyxovirus
• reverse genetic analysis
• ribozymes

• Calorimetry
• Circular Dichroism
• DNA, Intergenic
• Humans
• Mass Spectrometry
• Measles/metabolism
• Measles/virology
• Measles virus/chemistry
• Measles virus/metabolism
• Models, Molecular
• Nucleocapsid Proteins
• Nucleoproteins/chemistry
• Nucleoproteins/metabolism
• Protein Binding
• Transcription, Genetic
• Viral Proteins/chemistry
• Viral Proteins/metabolism
• Virus Replication/physiology

• agence nationale de la recherche
• Agence Nationale de la Recherche
• Fondation pour la Recherche Médicale (FR)

• Ebola virus
• Marburg virus
• biosafety level 4
• filoviruses
• full-length clones
• minigenome
• nonsegmented negative-sense RNA viruses
• reverse genetics system
• virus rescue
• virus-like particles

• Marburgvirus/genetics
• Marburgvirus/pathogenicity
• Marburgvirus/physiology
• Reverse Genetics/methods
• Virology/methods

• R03 AI114293 NIAID NIH HHS
• UC6 AI058618 NIAID NIH HHS

Reverse genetics systems have been established for all major groups of plant DNA and positive-strand RNA viruses, and our understanding of their infection cycles and pathogenesis has benefitted enormously from use of these approaches. However, technical difficulties have heretofore hampered applications of reverse genetics to plant negative-strand RNA (NSR) viruses. Here, we report recovery of infectious virus from cloned cDNAs of a model plant NSR, Sonchus yellow net rhabdovirus (SYNV). The procedure involves Agrobacterium-mediated transcription of full-length SYNV antigenomic RNA and co-expression of the nucleoprotein (N), phosphoprotein (P), large polymerase core proteins and viral suppressors of RNA silencing in Nicotiana benthamiana plants. Optimization of core protein expression resulted in up to 26% recombinant SYNV (rSYNV) infections of agroinfiltrated plants. A reporter virus, rSYNV-GFP, engineered by inserting a green fluorescence protein (GFP) gene between the N and P genes was able to express GFP during systemic infections and after repeated plant-to-plant mechanical passages. Deletion analyses with rSYNV-GFP demonstrated that SYNV cell-to-cell movement requires the sc4 protein and suggested that uncoiled nucleocapsids are infectious movement entities. Deletion analyses also showed that the glycoprotein is not required for systemic infection, although the glycoprotein mutant was defective in virion morphogenesis. Taken together, we have developed a robust reverse genetics system for SYNV that provides key insights into morphogenesis and movement of an enveloped plant virus. Our study also provides a template for developing analogous systems for reverse genetic analysis of other plant NSR viruses.

Reverse genetics is a powerful tool for fundamental studies of virus biology, pathology and biotechnology applications. Although plant negative-strand RNA (NSR) viruses consist of members in the Rhabdoviridae, Bunyaviridae, Ophioviridae families and several unassigned genera that collectively account for many economically important crop diseases, unfortunately, several technical difficulties have hindered application of genetic engineering to these groups of viruses. This study describes the first reverse genetics system developed for plant NSR viruses. We report an efficient procedure for production of infectious virus from cloned cDNAs of sonchus yellow net virus (SYNV) RNAs, a model plant rhabdovirus. We have also engineered a recombinant SYNV vector for stable expression of a fluorescent reporter gene. Using this system, we have generated targeted SYNV mutants whose analyses provide key insights into enveloped plant virus movement and morphogenesis processes. Moreover, our findings provide a template for reverse genetics studies with other plant rhabdoviruses, and a strategy to circumvent technical difficulties that have hampered these applications to plant NSR viruses.

• Ebola virus
• RNA editing site
• mutation
• reverse genetics system

• Attenuation
• Cancer therapy
• Innate immunity
• Mononegavirales
• Pathology
• Receptors
• Reverse genetics
• Tropism
• Vaccines

Negative-sense RNA viruses comprise several zoonotic pathogens that mutate rapidly and frequently emerge in people including Influenza, Ebola, Rabies, Hendra and Nipah viruses. Acute respiratory distress syndrome, encephalitis and vasculitis are common disease outcomes in people as a result of pathogenic viral infection, and are also associated with high case fatality rates. Viral spread from exposure sites to systemic tissues and organs is mediated by virulence factors, including viral attachment glycoproteins and accessory proteins, and their contribution to infection and disease have been delineated by reverse genetics; a molecular approach that enables researchers to experimentally produce recombinant and reassortant viruses from cloned cDNA. Through reverse genetics we have developed a deeper understanding of virulence factors key to disease causation thereby enabling development of targeted antiviral therapies and well-defined live attenuated vaccines. Despite the value of reverse genetics for virulence factor discovery, classical reverse genetic approaches may not provide sufficient resolution for characterization of heterogeneous viral populations, because current techniques recover clonal virus, representing a consensus sequence. In this review the contribution of reverse genetics to virulence factor characterization is outlined, while the limitation of the technique is discussed with reference to new technologies that may be utilized to improve reverse genetic approaches.

• Reverse genetics
• Viral pathogen
• Negative sense RNA viruses
• Influenza A virus
• Ebola virus
• Rabies virus
• Hendra virus
• Nipah virus

• Plant Leaves/metabolism
• Plant Leaves/virology
• Plant Viruses/genetics
• Plant Viruses/metabolism
• Plasmids
• RNA Viruses/genetics
• RNA Viruses/metabolism
• RNA, Plant/genetics
• Replicon
• Rhabdoviridae/physiology
• Rhabdoviridae Infections/genetics
• Rhabdoviridae Infections/virology
• Sonchus
• Nicotiana/genetics
• Nicotiana/metabolism
• Nicotiana/virology
• Transcription, Genetic
• Viral Proteins/genetics
• Viral Proteins/metabolism

• R03 AI059178 NIAID NIH HHS
• R03 AI059178-01 NIAID NIH HHS

• Antiviral Agents/pharmacology
• Arenaviridae/drug effects
• Arenaviridae/genetics
• Arenaviridae/physiology
• Bunyaviridae/drug effects
• Bunyaviridae/genetics
• Bunyaviridae/physiology
• DNA, Complementary/genetics
• DNA, Complementary/metabolism
• Filoviridae/drug effects
• Filoviridae/genetics
• Filoviridae/physiology
• Genes, Reporter
• Genome, Viral
• Ribonucleoproteins/genetics
• Ribonucleoproteins/metabolism
• Transcription, Genetic
• Transfection
• Viral Proteins/genetics
• Viral Proteins/metabolism
• Virus Internalization
• Virus Release
• Virus Replication

• Z99 AI999999 Intramural NIH HHS
• ZID AI009006-02 Intramural NIH HHS
• CIHR

Aquaculture has expanded rapidly to become a major economic and food-producing sector worldwide these last 30 years. In parallel, viral diseases have emerged and rapidly spread from farm to farm causing enormous economic losses. The most problematic viruses encountered in the field are mainly, but not exclusively, RNA viruses belonging to the Novirhabdovirus, Aquabirnavirus, Alphavirus and Betanodavirus genera. The recent establishment of reverse genetics systems to recover infectious fish RNA viruses entirely from cDNA has made possible to genetically manipulate the viral genome. These systems have provided powerful tools to study all aspects of the virus biology and virus-host interactions but also gave the opportunity to use these viruses as live vaccines or as gene vectors. This review provides an overview on the recent breakthroughs achieved by using these reverse genetics systems in terms of viral protein function, virulence and host-specificity factor, vaccine development and vector design.

• arenaviruses
• lassa virus
• junin virus
• lymphocytic choriomeningitis virus
• reverse genetics
• antivirals

• Animals
• Antiviral Agents/pharmacology
• Arenaviridae Infections/drug therapy
• Arenaviridae Infections/immunology
• Arenaviridae Infections/prevention & control
• Arenaviridae Infections/virology
• Arenavirus/drug effects
• Arenavirus/genetics
• Arenavirus/immunology
• Arenavirus/physiology
• Drug Evaluation, Preclinical
• Humans
• Promoter Regions, Genetic
• Viral Vaccines/genetics
• Viral Vaccines/immunology
• Virus Physiological Phenomena/drug effects

• R56 AI047140 NIAID NIH HHS
• U54 AI065359-040007 NIAID NIH HHS
• R01 AI047140-06A1 NIAID NIH HHS
• AI-065359 NIAID NIH HHS
• R01 AI047140 NIAID NIH HHS
• U54 AI065359 NIAID NIH HHS
• AI47140 NIAID NIH HHS

• paramyxovirus
• nipah virus
• rna polymerase i-driven minigenome rescue
• antiviral drug screening
• high throughput

• Antiviral Agents/pharmacology
• Cell Line
• Chloramphenicol O-Acetyltransferase/metabolism
• DNA-Directed RNA Polymerases/genetics
• DNA-Directed RNA Polymerases/metabolism
• Genome, Viral/genetics
• HeLa Cells
• Humans
• Microbial Sensitivity Tests/methods
• Nipah Virus/drug effects
• Nipah Virus/genetics
• Nipah Virus/physiology
• Paramyxoviridae/genetics
• Paramyxoviridae/metabolism
• Plasmids/genetics
• RNA Polymerase I/genetics
• RNA Polymerase I/metabolism
• RNA, Viral/drug effects
• RNA, Viral/genetics
• RNA, Viral/metabolism
• Transcription, Genetic/drug effects
• Transfection
• Viral Proteins/genetics
• Viral Proteins/metabolism
• Virus Replication/drug effects

• U54 AI057156 NIAID NIH HHS
• U54 AI057156-010009 NIAID NIH HHS

Embryonated chicken eggs have been used since the mid-20th century to grow a wide range of animal viruses to high titers. However, eggs have found so far only limited use in the production of recombinant proteins. We now describe a system, based on a Sendai virus minigenome, to produce large amounts of heterologous viral glycoproteins in the allantoic cavity of embryonated eggs.

Soluble forms of human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) fusion (F) proteins, devoid of their transmembrane and cytoplasmic domains, were produced in allantoic fluids using the Sendai minigenome system. The first step was rescuing in cell cultures Sendai virus minigenomes encoding the proteins of interest, with the help of wild type Sendai virus. The second step was propagating such recombinant defective viruses, together with the helper virus, in the allantoic cavity of chicken embryonated eggs, and passage to optimize protein production. When compared with the production of the same proteins in the culture supernatant of cells infected with vaccinia recombinants, the yield in the allantoic fluid was 5–10 fold higher. Mutant forms of these soluble proteins were easily constructed by site-directed mutagenesis and expressed in eggs using the same approach.

The simplicity and economy of the Sendai minigenome system, together with the high yield achieved in the allantoic fluid of eggs, makes it an attractive method to express soluble glycoproteins aimed for structural studies.

• Animals
• Cattle/virology
• Cattle Diseases/epidemiology
• Cattle Diseases/immunology
• Cattle Diseases/prevention & control
• Cattle Diseases/virology
• Respiratory Syncytial Virus Infections/epidemiology
• Respiratory Syncytial Virus Infections/prevention & control
• Respiratory Syncytial Virus Infections/veterinary
• Respiratory Syncytial Virus Infections/virology
• Respiratory Syncytial Virus, Bovine/genetics
• Respiratory Syncytial Virus, Bovine/immunology
• Respiratory Syncytial Virus, Bovine/pathogenicity
• Respiratory Syncytial Virus, Bovine/physiology

• Binding Sites
• Cell Nucleus/chemistry
• Cell Nucleus/virology
• Fluorescent Antibody Technique
• Karyopherins/metabolism
• Microscopy, Confocal
• Mutation
• Nuclear Localization Signals/genetics
• Nucleocapsid Proteins/genetics
• Nucleocapsid Proteins/metabolism
• Phosphoproteins/metabolism
• Plant Viruses/physiology
• Protein Binding
• Protein Structure, Tertiary
• Protein Transport
• Rhabdoviridae/physiology
• Sonchus/virology
• Two-Hybrid System Techniques
• Viral Proteins/metabolism

• ebola virus
• hemorrhagic fever
• marburg virus
• nonsegmented negative-sense RNA viruses
• nucleocapsid complex
• replication
• reverse genetics
• transcription

Nipah virus (NiV) is an emerging paramyxovirus distinguished by its ability to cause fatal disease in both animal and human hosts. Together with Hendra virus (HeV), they comprise the genus Henipavirus in the Paramyxoviridae family. NiV and HeV are also restricted to Biosafety Level-4 containment and this has hampered progress towards examining details of their replication and morphogenesis. Here, we have established recombinant expression systems to study NiV particle assembly and budding through the formation of virus-like particles (VLPs).

When expressed by recombinant Modified Vaccinia virus Ankara (rMVA) or plasmid transfection, individual NiV matrix (M), fusion (F) and attachment (G) proteins were all released into culture supernatants in a membrane-associated state as determined by sucrose density gradient flotation and immunoprecipitation. However, co-expression of F and G along with M revealed a shift in their distribution across the gradient, indicating association with M in VLPs. Protein release was also altered depending on the context of viral proteins being expressed, with F, G and nucleocapsid (N) protein reducing M release, and N release dependent on the co-expression of M. Immunoelectron microscopy and density analysis revealed VLPs that were similar to authentic virus. Differences in the budding dynamics of NiV proteins were also noted between rMVA and plasmid based strategies, suggesting that over-expression by poxvirus may not be appropriate for studying the details of recombinant virus particle assembly and release.

Taken together, the results indicate that NiV M, F, and G each possess some ability to bud from expressing cells, and that co-expression of these viral proteins results in a more organized budding process with M playing a central role. These findings will aid our understanding of paramyxovirus particle assembly in general and could help facilitate the development of a novel vaccine approach for henipaviruses.

• Animals
• Cell Line
• Chick Embryo
• Chlorocebus aethiops
• Humans
• Nipah Virus/metabolism
• Nucleocapsid Proteins/metabolism
• Plasmids/genetics
• Recombination, Genetic
• Transfection
• Vaccinia virus/genetics
• Vaccinia virus/metabolism
• Vero Cells
• Viral Envelope Proteins/metabolism
• Viral Fusion Proteins/metabolism
• Viral Matrix Proteins/metabolism
• Virion/metabolism
• Virus Assembly

• R01 AI054715 NIAID NIH HHS
• U54 AI057168 NIAID NIH HHS
• AI054715 NIAID NIH HHS
• AI057168 NIAID NIH HHS