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At least 73 records · Page 4Linked to original sources
PubMed2005

Novel insights into the regulation of the viral polymerase complex of neurotropic Borna disease virus.

Borna disease virus (BDV) genetic information is encoded in a highly condensed non-segmented RNA genome of negative polarity. Replication and transcription of the genome occurs in the nucleus, enabling the virus to employ the cellular splicing machinery to process primary transcripts and to regulate expression of viral gene products. BDV establishes a non-cytolytic, persistent infection that in animals is mainly restricted to neurons of the central nervous system. Based on these unique properties, BDV represents the prototype member of the virus family Bornaviridae in the order Mononegavirales. Analysis of molecular aspects of BDV replication has long been hampered by the lack of a reverse genetics system. Only recently, artificial BDV minigenomes permitted the reconstitution of the viral polymerase complex, allowing finally the recovery of BDV from cDNA. As in other families of the Mononegavirales, the active polymerase complex of BDV is composed of the polymerase (L), the nucleoprotein (N) and the phosphoprotein (P). In addition, the viral X protein was identified as potent negative regulator of polymerase activity. Protein interaction studies combined with minireplicon assays suggested that P is a central regulatory element of BDV replication that directs the assembly of the polymerase complex. Most intriguingly, BDV obtained from cDNA with variable genomic termini suggests a novel strategy for viral replication-control. BDV seems to restrict its propagation efficacy by defined 5' terminal trimming of genomic and antigenomic RNA molecules. This review will summarize these novel findings and will discuss them in the context of BDV neurotropism and persistence.

Animals↗
PubMed1998

Transfectant influenza A viruses with long deletions in the NS1 protein grow efficiently in Vero cells.

We established a reverse genetics system for the nonstructural (NS) gene segment of influenza A virus. This system is based on the use of the temperature-sensitive (ts) reassortant virus 25A-1. The 25A-1 virus contains the NS gene from influenza A/Leningrad/134/57 virus and the remaining gene segments from A/Puerto Rico (PR)/8/34 virus. This particular gene constellation was found to be responsible for the ts phenotype. For reverse genetics of the NS gene, a plasmid-derived NS gene from influenza A/PR/8/34 virus was ribonucleoprotein transfected into cells that were previously infected with the 25A-1 virus. Two subsequent passages of the transfection supernatant at 40 degreesC selected viruses containing the transfected NS gene derived from A/PR/8/34 virus. The high efficiency of the selection process permitted the rescue of transfectant viruses with large deletions of the C-terminal part of the NS1 protein. Viable transfectant viruses containing the N-terminal 124, 80, or 38 amino acids of the NS1 protein were obtained. Whereas all deletion mutants grew to high titers in Vero cells, growth on Madin-Darby canine kidney (MDCK) cells and replication in mice decreased with increasing length of the deletions. In Vero cells expression levels of viral proteins of the deletion mutants were similar to those of the wild type. In contrast, in MDCK cells the level of the M1 protein was significantly reduced for the deletion mutants.

Animals↗
PubMed2000

Unidirectional RNA polymerase I-polymerase II transcription system for the generation of influenza A virus from eight plasmids.

Recently, we developed a system for the generation of influenza A virus by cotransfecting only eight plasmids from which negative-sense vRNA and positive-sense mRNA are expressed (Hoffmann et al., Proceedings of the National Academy of Sciences, USA 97, 6108-6113, 2000). Here we report the establishment of a different transcription system for the expression of virus-like RNAs, allowing the intracellular synthesis of noncapped positive-sense cRNA and 5'-capped mRNA from one template. Cotransfection of eight RNA pol I-pol II tandem promoter plasmids containing the cDNA of A/WSN/33 (H1N1) resulted in the generation of infectious influenza A virus, albeit with a lower yield than the bidirectional system. Our approach of producing either vRNA and mRNA or cRNA and mRNA intracellularly from a minimum set of plasmids should be useful for the establishment or optimization of reverse genetics systems for other RNA viruses.

Animals↗
PubMed2006

Betanodavirus infection in the freshwater model fish medaka (Oryzias latipes).

Betanodaviruses, the causal agents of viral nervous necrosis in marine fish, have bipartite, positive-sense RNA genomes. As their genomes are the smallest and simplest among viruses, betanodaviruses have been studied in detail as model viruses by using a genetic-engineering system, as has occurred with the insect alphanodaviruses, the other members of the family Nodaviridae. However, studies of virus-host interactions have been limited, as betanodaviruses basically infect marine fish at early developmental stages (larval and juvenile). These fish are only available for a few months of the year and are not suitable for the construction of a reverse-genetics system. To overcome these problems, several freshwater fish species were tested for their susceptibility to betanodaviruses. It was found that adult medaka (Oryzias latipes), a well-known model fish, was susceptible to both Striped jack nervous necrosis virus (the type species of the genus Betanodavirus) and Redspotted grouper nervous necrosis virus (RGNNV), which have different host specificities in marine fish species. Infected medaka exhibited erratic swimming and the viruses were localized specifically in the brain, spinal cord and retina of the infected fish, similar to the pattern of infection in naturally infected marine fish. Moreover, medaka were susceptible to RGNNV at the larval stage. This is the first report of a model virus-model host infection system in fish. This system should facilitate elucidation of the mechanisms underlying RNA virus infections in fish.

Animals↗
PubMed2006

Establishment of a Nipah virus rescue system.

Nipah virus (NiV), a paramyxovirus, was first discovered in Malaysia in 1998 in an outbreak of infection in pigs and humans and incurred a high fatality rate in humans. Fruit bats, living in vast areas extending from India to the western Pacific, were identified as the natural reservoir of the virus. However, the mechanisms that resulted in severe pathogenicity in humans (up to 70% mortality) and that enabled crossing the species barrier were not known. In this study, we established a system that enabled the rescue of replicating NiVs from a cloned DNA by cotransfection of a constructed full-length cDNA clone and supporting plasmids coding virus nucleoprotein, phosphoprotein, and polymerase with the infection of the recombinant vaccinia virus, MVAGKT7, expressing T7 RNA polymerase. The rescued NiV (rNiV), by using the newly developed reverse genetics system, showed properties in vitro that were similar to the parent virus and retained the severe pathogenicity in a previously established animal model by experimental infection. A recombinant NiV was also developed, expressing enhanced green fluorescent protein (rNiV-EGFP). Using the virus, permissibility of NiV was compared with the presence of a known cellular receptor, ephrin B2, in a number of cell lines of different origins. Interestingly, two cell lines expressing ephrin B2 were not susceptible for rNiV-EGFP, indicating that additional factors are clearly required for full NiV replication. The reverse genetics for NiV will provide a powerful tool for the analysis of the molecular mechanisms of pathogenicity and cross-species infection.

Animals↗
PubMed2001

Functional analysis of PA binding by influenza a virus PB1: effects on polymerase activity and viral infectivity.

Influenza A virus expresses three viral polymerase (P) subunits-PB1, PB2, and PA-all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain alpha, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain alpha. The role of the minimal PB1 domain alpha in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

Amino Acid Sequence↗
PubMed2001

Rescue of recombinant Thogoto virus from cloned cDNA.

Thogoto virus (THOV) is a tick-transmitted orthomyxovirus with a genome consisting of six negative-stranded RNA segments. To rescue a recombinant THOV, the viral structural proteins were produced from expression plasmids by means of a vaccinia virus expressing the T7 RNA polymerase. Genomic virus RNAs (vRNAs) were generated from plasmids under the control of the RNA polymerase I promoter. Using this system, we could efficiently recover recombinant THOV following transfection of 12 plasmids into 293T cells. To verify the recombinant nature of the rescued virus, specific genetic tags were introduced into two vRNA segments. The availability of this efficient reverse genetics system will allow us to address hitherto-unanswered questions regarding the biology of THOV by manipulating viral genes in the context of infectious virus.

Animals↗
PubMed1999

Generation of influenza A viruses entirely from cloned cDNAs.

We describe a new reverse-genetics system that allows one to efficiently generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding a viral RNA of the A/WSN/33 (H1N1) or A/PR/8/34 (H1N1) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator-together with plasmids encoding viral nucleoprotein and the PB2, PB1, and PA viral polymerases. This strategy yielded >1 x 10(3) plaque-forming units (pfu) of virus per ml of supernatant at 48 hr posttransfection. The addition of plasmids expressing all of the remaining viral structural proteins led to a substantial increase in virus production, 3 x 10(4)-5 x 10(7) pfu/ml. We also used reverse genetics to generate a reassortant virus containing the PB1 gene of the A/PR/8/34 virus, with all other genes representing A/WSN/33. Additional viruses produced by this method had mutations in the PA gene or possessed a foreign epitope in the head of the neuraminidase protein. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and in the production of vaccines and gene therapy vectors.

Animals↗
PubMed2002

A decade after the generation of a negative-sense RNA virus from cloned cDNA - what have we learned?

Since the first generation of a negative-sense RNA virus entirely from cloned cDNA in 1994, similar reverse genetics systems have been established for members of most genera of the Rhabdo- and Paramyxoviridae families, as well as for Ebola virus (Filoviridae). The generation of segmented negative-sense RNA viruses was technically more challenging and has lagged behind the recovery of nonsegmented viruses, primarily because of the difficulty of providing more than one genomic RNA segment. A member of the Bunyaviridae family (whose genome is composed of three RNA segments) was first generated from cloned cDNA in 1996, followed in 1999 by the production of influenza virus, which contains eight RNA segments. Thus, reverse genetics, or the de novo synthesis of negative-sense RNA viruses from cloned cDNA, has become a reliable laboratory method that can be used to study this large group of medically and economically important viruses. It provides a powerful tool for dissecting the virus life cycle, virus assembly, the role of viral proteins in pathogenicity and the interplay of viral proteins with components of the host cell immune response. Finally, reverse genetics has opened the way to develop live attenuated virus vaccines and vaccine vectors.

Animals↗
PubMed2001

Universal primer set for the full-length amplification of all influenza A viruses.

To systematically identify and analyze the 15 HA and 9 NA subtypes of influenza A virus, we need reliable, simple methods that not only characterize partial sequences but analyze the entire influenza A genome. We designed primers based on the fact that the 15 and 21 terminal segment specific nucleotides of the genomic viral RNA are conserved between all influenza A viruses and unique for each segment. The primers designed for each segment contain influenza virus specific nucleotides at their 3'-end and non-influenza virus nucleotides at the 5'-end. With this set of primers, we were able to amplify all eight segments of N1, N2, N4, N5, and N8 subtypes. For N3, N6, N7, and N9 subtypes, the segment specific sequences of the neuraminidase genes are different. Therefore, we optimized the primer design to allow the amplification of those neuraminidase genes as well. The resultant primer set is suitable for all influenza A viruses to generate full-length cDNAs, to subtype viruses, to sequence their DNA, and to construct expression plasmids for reverse genetics systems.

Child↗
PubMed2006

The 3' sequences required for incorporation of an engineered ssRNA into the Reovirus genome.

BACKGROUND: Understanding how an organism replicates and assembles a multi-segmented genome with fidelity previously measured at 100% presents a model system for exploring questions involving genome assortment and RNA/protein interactions in general. The virus family Reoviridae, containing nine genera and more than 200 members, are unique in that they possess a segmented double-stranded (ds) RNA genome. Using reovirus as a model member of this family, we have developed the only functional reverse genetics system for a member of this family with ten or more genome segments. Using this system, we have previously identified the flanking 5' sequences required by an engineered s2 ssRNA for efficient incorporation into the genome of reovirus. The minimum 5' sequence retains 96 nucleotides and contains a predicted sequence/structure element. Within these 96 nucleotides, we have identified three nucleotides A-U-U at positions 79-81 that are essential for the incorporation of in vitro generated ssRNAs into new reovirus progeny viral particles. The work presented here builds on these findings and presents the results of an analysis of the required 3' flanking sequences of the s2 ssRNA. RESULTS: The minimum 3' sequence we localized retains 98 nucleotides of the wild type s2 ssRNA. These sequences do not interact with the 5' sequences and modifications of the 5' sequences does not result in a change in the sequences required at the 3' end of the engineered s2 ssRNA. Within the 3' sequence we discovered three regions that when mutated prevent the ssRNA from being replicated to dsRNA and subsequently incorporated into progeny virions. Using a series of substitutions we were able to obtain additional information about the sequences in these regions. We demonstrate that the individual nucleotides from, 98 to 84, 68 to 59, and 28 to 1, are required in addition to the total length of 98 nucleotides to direct an engineered reovirus ssRNA to be replicated to dsRNA and incorporated into a progeny virion. Extensive analysis using a number of RNA structure-predication software programs revealed three possible structures predicted to occur in all 10 reovirus ssRNAs but not predicted to contain conserved individual nucleotides that we could probe further by using individual nucleotide substitutions. The presence of a conserved structure would permit all ten ssRNAs to be identified and selected as a set, while unique nucleotides within the structure would direct the set to contain 10 unique members. CONCLUSION: This study completes the characterization and mapping of the 5' and 3' sequences required for an engineered reovirus s2 ssRNA to be incorporated into an infectious progeny virus and establishes a firm foundation for additional investigations into the assortment and encapsidation mechanism of all 10 ssRNAs into the dsRNA genome of reovirus. As researchers build on this work and apply this system to additional reovirus genes and additional dsRNA viruses, a complete model for genome assortment and replication for these viruses will emerge.

Animals↗
PubMed2004

Severe acute respiratory syndrome coronavirus pathogenesis, disease and vaccines: an update.

BACKGROUND: A novel coronavirus has recently been identified as the cause of severe acute respiratory syndrome (SARS-CoV). The ability of this family of positive strand RNA viruses to move between species and cause severe disease in humans, with the potential for pandemic spread, has been confirmed. METHODS: An understanding of the disease and its pathogenesis and the genetics of coronavirus infections, as well as strategies to treat or prevent coronavirus infections, are essential. The history of coronavirus vaccines and the occurrence of laboratory-associated SARS-CoV infections underscore the need for stably attenuated strains of SARS-CoV and other coronaviruses. RESULTS: Rapid progress has been made in understanding the clinical disease of SARS in adults and children. In adults, systemic infection with clinical and biochemical abnormalities, as well as respiratory infection, may be the rule. SARS is much milder in children younger than 12 years old than it is in adolescents and adults. In children age 12 years and younger, symptoms are generally nonspecific and cold-like. Numerous approaches to the development of SARS-CoV vaccines have been undertaken, and there is evidence that antibodies to the spike protein may be protective from replication and pathology in animal models. CONCLUSIONS: The availability of reverse genetic systems has made it possible to engineer and recover coronavirus variants that contain multiple genetically stable mutations that grow well in culture but are attenuated for replication, virulence or both. Such variants will be platforms for the safe growth of SARS-CoV and candidates for live attenuated vaccines.

Adolescent↗
PubMed2000

"Ambisense" approach for the generation of influenza A virus: vRNA and mRNA synthesis from one template.

We present a system for creating influenza virus by generating viral RNA (vRNA) and mRNA from one template. Recently, a system for the generation of influenza A virus entirely from cloned cDNAs was established (Neumann et al., 1999, Proc. Natl. Acad. Sci. USA 96, 9345-9350). Cells were transfected with plasmids for RNA polymerase I-driven intracellular synthesis of all eight viral RNAs, and with protein expression plasmids for the synthesis of viral structural proteins. Although this system is highly efficient in virus generation, the construction and cotransfection of 17 plasmids is cumbersome and may limit the use of this system to cell lines that can be transfected with high efficiencies. Synthesizing both vRNA and mRNA from one template would reduce the number of plasmids required for virus generation. Therefore, we generated a bidirectional transcription construct that contains cDNA encoding PB1 flanked by an RNA polymerase I (pol I) promoter for vRNA synthesis and an RNA polymerase II (pol II) promoter for mRNA synthesis. The utility of this approach is proved by the generation of virus after transfecting the pol I/pol II-promoter-PB1 construct together with vRNA- and protein-expression constructs for the remaining seven segments. Because this approach reduces the number of plasmids required for virus generation, it also reduces the work necessary for cloning, probably enhances the efficiency of virus generation, and expands the use of the reverse-genetics system to cell lines for which efficient cotransfection of 17 plasmids cannot be achieved.

Animals↗
PubMed2001

Complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains.

Two full-length porcine reproductive and respiratory syndrome virus (PRRSV) genomes, strain VR-2332 and its cell culture passaged descendent RespPRRS vaccine strain, were compared and analyzed in order to identify possible sites of attenuation. Of the 44 nucleotide changes, 13 resulted in conservative changes and 18 produced non-conservative changes. The results suggest that key amino acids in ORF1 may contribute to the phenotype of RespPRRS, which includes increased growth rate on MA-104 cells and decreased virulence in swine. The results provide a genetic basis for future manipulation of a PRRSV reverse genetics system.

5' Untranslated Regions↗
PubMed2003

A ferret model of canine distemper virus virulence and immunosuppression.

Canine distemper virus (CDV) infects many carnivores, including ferrets and dogs, and is the member of the Morbillivirus genus most easily amenable to experimentation in a homologous small-animal system. To gain insights into the determinants of CDV pathogenesis, we isolated a strain highly virulent for ferrets by repeated passaging in these animals. Sequence comparison of the genome of this strain with that of its highly attenuated precursor revealed 19 mutations distributed almost evenly in the six genes. We then recovered a virus from a cDNA copy of the virulent CDV strain's consensus sequence by using a modified reverse genetics system based on B cells. We infected ferrets with this virus and showed that it fully retained virulence as measured by the timing of rash appearance, disease onset, and death. Body temperature, leukocyte number, lymphocyte proliferation activity, and cell-associated viremia also had similar kinetics. We then addressed the question of the relative importance of the envelope and other viral constituents for virulence. Viruses in which the envelope genes (matrix, fusion, and hemagglutinin) of the virulent strain were combined with the other genes of the attenuated strain caused severe rash and fever even if the disease onset was delayed. Viruses in which the nucleocapsid, polymerase, and phosphoprotein genes (coding also for the V and C proteins) of the virulent strain were combined with the envelope genes of the attenuated strain caused milder signs of disease. Thus, virulence-inducing mutations have accumulated throughout the genome.

Animals↗
PubMed2004

Generation of reassortant influenza vaccines by reverse genetics that allows utilization of a DIVA (Differentiating Infected from Vaccinated Animals) strategy for the control of avian influenza.

Vaccination of poultry with inactivated influenza vaccine can be an effective tool in the control of avian influenza (AI). One major concern of using inactivated vaccine is vaccine-induced antibody interference with serologic surveillance and epidemiology. In the United States, low pathogenicity H5 and H7 subtype AI viruses have caused serious economic losses in the poultry industry. Most of these viruses also have the accompanying N2 subtype and no H5N1 or H7N8 subtype AI viruses have been identified in poultry in the US. In order to allow the Differentiation of Infected from Vaccinated Animals (DIVA) while maintaining maximum efficacy of the vaccine, we generated reassortant viruses by reverse genetics that contained the same H5 and H7 hemagglutinin (HA) gene as the challenge virus, but a heterologous N1 or N8 neuraminidase (NA) gene. In vaccination-challenge experiments in 2-week-old specific pathogen free chickens, reassortant influenza vaccines (rH5N1 and rH7N8) demonstrated similar antibody profiles and comparable protection rates as vaccines prepared with parent H5N2 and H7N2 viruses. Further, we were able to differentiate the sera from infected and vaccinated birds by neuraminidase inhibition test and indirect immunofluorescent antibody assay on the basis of different antibodies elicited by their NA proteins. These results demonstrate the usefulness of a reverse genetics system for the rapid generation of reassortant AI virus that allows utilization of the DIVA strategy for the control of AI infections in poultry.

Animals↗
PubMed1997

Authentic and chimeric full-length genomic cDNA clones of bovine viral diarrhea virus that yield infectious transcripts.

Bovine viral diarrhea virus (BVDV) is the most insidious and devastating viral pathogen of cattle in the United States. Disease control approaches must be based on detailed knowledge of virus biology. To develop reverse-genetic systems to study the molecular biology of the virus, we first constructed a plasmid containing the entire genome of BVDV cloned as cDNA. Subsequently, we showed that infectious BVDV was produced by cells transfected with uncapped RNA transcribed in vitro from the cDNA clone. This result defined functional 5' and 3' termini in viral genomic RNA and established the biological importance of the proposed internal ribosome entry site element in the 5' untranslated region of the viral genome. BVDV rescued from the infectious cDNA clone has an in vitro phenotype similar to that of the wild-type parent, the National Animal Disease Laboratory strain of BVDV. A deletion of a single codon in the full-length genomic BVDV cDNA clone, encoding glutamic acid at position 1600, gave rise to sequence-tagged virus easily identified by restriction fragment length polymorphism analysis of reverse transcription-PCR amplicons. Suitability of the molecular clone of BVDV for genomic manipulations was shown by substitution of the major envelope glycoprotein E2/gp53 with that of the Singer strain, giving rise to a chimeric virus. The predicted change in antigenic structure of the chimeric virus could be readily identified with strain-specific monoclonal antibodies by neutralization and immunofluorescence assays. Immediate applications of this system include development of safe and effective live vaccine strains possessing predetermined defined attenuating mutations.

Animals↗
PubMed2006

Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis.

The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2'-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.

Animals↗