Harmonizing Global Biospecimen Consent Practices to Advance Translational Research: A Call to Action.

One of the many challenges of translational medicine is working with research participants to donate biospecimens through an ethical informed consent framework. The increasingly complex ethical and regulatory differences across jurisdictions translates into limitations on use and potential value of biological specimens and their associated data in clinical research. We introduce a call to action for more uniform global standards for collection of biological specimen informed consent data to enable greater advancements in medical research.

Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2008).

In accordance with the Statutes of the International Committee of Taxonomy of Viruses (ICTV), the final stage in the process of making changes to the Universal Scheme of Virus Classification is the ratification of taxonomic proposals by ICTV Members. This can occur either at a Plenary meeting of ICTV, held during an International Congress of Virology meeting, or by circulation of proposals by mail followed by a ballot. Therefore, a list of proposals that had been subjected to the full, multi-stage review process was prepared and presented on the ICTVonline web pages in March 2008. This review process involved input from the ICTV Study Groups and Subcommittees, other interested virologists, and the ICTV Executive Committee. For the first time, the ratification process was performed entirely by email. The proposals were sent electronically via email on 18 March 2008 to ICTV Life Members (11), ICTV Subcommittee Members (74), and ICTV National Representatives (53).

Preliminary x-ray characterization of authentic providence virus and attempts to express its coat protein gene in recombinant baculovirus.

Providence Virus (PrV) is a non-envoloped, T = 4 icosahedral beta-tetravirus that undergoes autocatalytic cleavage of its coat protein precursor after capsid assembly. This is also a well characterized function of Nudaurelia capensis omega virus (NomegaV), a member of the related omegatetraviruses, whose x-ray structure has been determined. Virus-like particle (VLP) production of PrV in a recombinant baculovirus expression system was attempted to obtain high VLP yields for comparison of structural and autocatalytic active site properties between these virus groups. This resulted in insoluble aggregates of PrV coat protein even though NomegaV VLPs have been successfully produced in the same system. Betatetraviruses may be more dependent on compartmentalization and availability of their full-length genome for proper folding and assembly. However, crystals were grown of limited quantities of authentic PrV produced in cell culture and a partial X-ray data set collected to 3.8 A resolution. The virus particle position and orientation in the unit cell was determined by space group consideration and rotation function analysis. A phasing model, based on NomegaV, was developed to initiate the structure solution of PrV.

DNA-directed expression of an animal virus RNA for replication-dependent colony formation in Saccharomyces cerevisiae.

To date, the insect nodavirus flock house virus (FHV) is the only virus of a higher eukaryote that has been shown to undergo a full replicative cycle and produce infectious progeny in the yeast Saccharomyces cerevisiae. The genome of FHV is composed of two positive-sense RNA segments: RNA1, encoding the RNA replicase, and RNA2, encoding the capsid protein precursor. When yeast cells expressing FHV RNA replicase were transfected with a chimeric RNA composed of a selectable gene flanked by the termini of RNA2, the chimeric RNA was replicated and transmitted to daughter cells indefinitely. In the work reported here, we developed a system in which a selectable chimeric RNA replicon was transcribed from an inducible RNA polymerase II (polII) promoter in vivo in yeast. To render marker gene expression absolutely dependent on RNA replication, the primary polII transcript was made negative in sense and contained an intron that blocked the translation of cryptic transcripts from the opposite DNA strand. The RNA products of DNA-templated transcription, processing, and RNA replication were characterized by Northern blot hybridization and primer extension analysis. Marker gene expression and colony growth under selective conditions depended strictly on FHV RNA replication, with background colonies arising at a frequency of fewer than 1 in 10(8) plated cells. The utility of the system was demonstrated by introducing a second chimeric replicon and showing that at least two different selectable markers could be simultaneously expressed by means of RNA replication. This is the first example of FHV RNA1-dependent selectable marker expression initiated in vivo and will greatly facilitate the identification and characterization of the requirements and inhibitors of RNA replication.

Characterization and template properties of RNA dimers generated during flock house virus RNA replication.

Flock house virus (FHV) is the best studied member of the Nodaviridae, a family of small, nonenveloped, isometric RNA viruses of insects and fish. Nodavirus genomes comprise two single-stranded positive-sense RNA segments (RNAs 1 and 2) that encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively. The RdRp replicates both genomic RNAs and also generates a subgenomic RNA (RNA3) that is not encapsidated. Although genomic RNAs replicate through negative-sense intermediates, little is known about these RNAs or the details of the replication mechanism. Negative-sense RNAs 1, 2, and 3, as well as putative dimers of RNAs 2 and 3, have been detected in previous studies. In this study we detected dimers of RNAs 1, 2, and 3 by Northern blot analyses of RNA samples from FHV-infected Drosophila cells, as well as from mammalian and yeast cells supporting FHV RNA replication. Characterization of these RNA species by RT-PCR and sequence determination showed that they contained head-to-tail junctions of FHV RNAs. RNAs containing the complete sequence of RNA2 joined to RNA3 were also detected during replication. To examine the template properties of these dimeric RNAs, we made corresponding cDNAs and transcribed them from a T7 promoter in mammalian cells constitutively expressing T7 RNA polymerase, together with RNA1 to provide the RdRp. Although heterologous terminal extensions inhibit FHV RNA replication, monomeric RNA2 was resolved and replicated from complete or partial homodimer templates and from an RNA2-RNA3 heterodimer.

Recovery of infectious pariacoto virus from cDNA clones and identification of susceptible cell lines.

Pariacoto virus (PaV) is a nodavirus that was recently isolated in Peru from the Southern armyworm, Spodoptera eridania. Virus particles are non enveloped and about 30 nm in diameter and have T=3 icosahedral symmetry. The 3.0-A crystal structure shows that about 35% of the genomic RNA is icosahedrally ordered, with the RNA forming a dodecahedral cage of 25-nucleotide (nt) duplexes that underlie the inner surface of the capsid. The PaV genome comprises two single-stranded, positive-sense RNAs: RNA1 (3,011 nt), which encodes the 108-kDa catalytic subunit of the RNA-dependent RNA polymerase, and RNA2 (1,311 nt), which encodes the 43-kDa capsid protein precursor alpha. In order to apply molecular genetics to the structure and assembly of PaV, we identified susceptible cell lines and developed a reverse genetic system for this virus. Cell lines that were susceptible to infection by PaV included those from Spodoptera exigua, Helicoverpa zea and Aedes albopictus, whereas cells from Drosophila melanogaster and Spodoptera frugiperda were refractory to infection. To recover virus from molecular clones, full-length cDNAs of PaV RNAs 1 and 2 were cotranscribed by T7 RNA polymerase in baby hamster kidney cells that expressed T7 RNA polymerase. Lysates of these cells were infectious both for cultured cells from Helicoverpa zea (corn earworm) and for larvae of Galleria mellonella (greater wax moth). The combination of infectious cDNA clones, cell culture infectivity, and the ability to produce milligram amounts of virus allows the application of DNA-based genetic methods to the study of PaV structure and assembly.

Rearrangement of the genes of vesicular stomatitis virus eliminates clinical disease in the natural host: new strategy for vaccine development.

Gene expression among the nonsegmented negative-strand RNA viruses is controlled by distance from the single transcriptional promoter, so the phenotypes of these viruses can be systematically manipulated by gene rearrangement. We examined the potential of gene rearrangement as a means to develop live attenuated vaccine candidates against Vesicular stomatitis virus (VSV) in domestic swine, a natural host for this virus. The results showed that moving the nucleocapsid protein gene away from the single transcriptional promoter attenuated and ultimately eliminated the potential of the virus to cause disease. Combining this change with relocation of the surface glycoprotein gene yielded a vaccine that protected against challenge with wild-type VSV. By incremental manipulation of viral properties, gene rearrangement provides a new approach to generating live attenuated vaccines against this class of virus.

The structure of pariacoto virus reveals a dodecahedral cage of duplex RNA.

The 3.0 A resolution crystal structure of Pariacoto virus (PaV) reveals extensive interactions between portions of the viral RNA genome and the icosahedral capsid. Under the protein shell of the T = 3 quasi equivalent capsid lies a dodecahedral cage composed of RNA duplex that accounts for approximately 35% of the single-stranded RNA genome. The highly basic N-terminal regions (residues 7-54) of the subunits, forming pentamers (A subunits) are clearly visible in the density map and make numerous interactions with the RNA cage. The C-terminal segments (residues 394-401) of the A subunits lie in channels near the quasi three-fold axes. Electron cryo-microscopy and image reconstruction of PaV particles clearly show the dodecahedral RNA cage.

Moving the glycoprotein gene of vesicular stomatitis virus to promoter-proximal positions accelerates and enhances the protective immune response.

Vesicular stomatitis virus (VSV) is the prototype of the Rhabdoviridae and contains nonsegmented negative-sense RNA as its genome. The 11-kb genome encodes five genes in the order 3'-N-P-M-G-L-5', and transcription is obligatorily sequential from the single 3' promoter. As a result, genes at promoter-proximal positions are transcribed at higher levels than those at promoter-distal positions. Previous work demonstrated that moving the gene encoding the nucleocapsid protein N to successively more promoter-distal positions resulted in stepwise attenuation of replication and lethality for mice. In the present study we investigated whether moving the gene for the attachment glycoprotein G, which encodes the major neutralizing epitopes, from its fourth position up to first in the gene order would increase G protein expression in cells and alter the immune response in inoculated animals. In addition to moving the G gene alone, we also constructed viruses having both the G and N genes rearranged. This produced three variant viruses having the orders 3'-G-N-P-M-L-5' (G1N2), 3'-P-M-G-N-L-5' (G3N4), and 3'-G-P-M-N-L-5' (G1N4), respectively. These viruses differed from one another and from wild-type virus in their levels of gene expression and replication in cell culture. The viruses also differed in their pathogenesis, immunogenicity, and level of protection of mice against challenge with wild-type VSV. Translocation of the G gene altered the kinetics and level of the antibody response in mice, and simultaneous reduction of N protein expression reduced replication and lethality for animals. These studies demonstrate that gene rearrangement can be exploited to design nonsegmented negative-sense RNA viruses that have characteristics desirable in candidates for live attenuated vaccines.

Characterization and construction of functional cDNA clones of Pariacoto virus, the first Alphanodavirus isolated outside Australasia.

Pariacoto virus (PaV) was recently isolated in Peru from the Southern armyworm (Spodoptera eridania). PaV particles are isometric, nonenveloped, and about 30 nm in diameter. The virus has a bipartite RNA genome and a single major capsid protein with a molecular mass of 39.0 kDa, features that support its classification as a Nodavirus. As such, PaV is the first Alphanodavirus to have been isolated from outside Australasia. Here we report that PaV replicates in wax moth larvae and that PaV genomic RNAs replicate when transfected into cultured baby hamster kidney cells. The complete nucleotide sequences of both segments of the bipartite RNA genome were determined. The larger genome segment, RNA1, is 3,011 nucleotides long and contains a 973-amino-acid open reading frame (ORF) encoding protein A, the viral contribution to the RNA replicase. During replication, a 414-nucleotide long subgenomic RNA (RNA3) is synthesized which is coterminal with the 3' end of RNA1. RNA3 contains a small ORF which could encode a protein of 90 amino acids similar to the B2 protein of other alphanodaviruses. RNA2 contains 1,311 nucleotides and encodes the 401 amino acids of the capsid protein precursor alpha. The amino acid sequences of the PaV capsid protein and the replicase subunit share 41 and 26% identity with homologous proteins of Flock house virus, the best characterized of the alphanodaviruses. These and other sequence comparisons indicate that PaV is evolutionarily the most distant of the alphanodaviruses described to date, consistent with its novel geographic origin. Although the PaV capsid precursor is cleaved into the two mature capsid proteins beta and gamma, the amino acid sequence at the cleavage site, which is Asn/Ala in all other alphanodaviruses, is Asn/Ser in PaV. To facilitate the investigation of PaV replication in cultured cells, we constructed plasmids that transcribed full-length PaV RNAs with authentic 5' and 3' termini. Transcription of these plasmids in cells recreated the replication of PaV RNA1 and RNA2, synthesis of subgenomic RNA3, and translation of viral proteins A and alpha.

Induction and maintenance of autonomous flock house virus RNA1 replication.

The nodavirus flock house virus (FHV) has a bipartite, positive-sense, RNA genome that encodes the catalytic subunit of the RNA replicase and the viral capsid protein precursor on separate genomic segments (RNA1 and RNA2, respectively). RNA1 can replicate autonomously when transfected into permissive cells, allowing study of the kinetics of RNA1 replication in the absence of either RNA2 or capsid proteins. However, RNA1 replication ceases ca. 3 days after transfection despite the presence of replication-competent RNA. We examined this inhibition by inducing the expression of RNA1 in cells from a cDNA copy that was under the control of a hormone-regulated RNA polymerase II promoter. This system reproduced the shutoff of RNA replication when DNA-templated primary transcription was turned off. Continued primary transcription partially alleviated the shutoff and maintained the rate of RNA replication for several days at a steady-state level approximately one-third that of the peak rate. After shutoff, RNA replication could be restored by transferring the resulting intracellular RNA to fresh cells or by reinducing primary transcription, indicating that cessation of replication occurred despite the competence of both the viral RNA and the cytoplasmic environment. These data suggest that there is a mechanism by which replication is shut off at late times after transfection, which may reflect the natural endpoint of the replicative cycle.

Phenotypic consequences of rearranging the P, M, and G genes of vesicular stomatitis virus.

The nonsegmented negative-strand RNA viruses (order Mononegavirales) include many important human pathogens. The order of their genes, which is highly conserved, is the major determinant of the relative levels of gene expression, since genes that are close to the single promoter site at the 3' end of the viral genome are transcribed at higher levels than those that occupy more distal positions. We manipulated an infectious cDNA clone of the prototypic vesicular stomatitis virus (VSV) to rearrange three of the five viral genes, using an approach which left the viral nucleotide sequence otherwise unaltered. The central three genes in the gene order, which encode the phosphoprotein P, the matrix protein M, and the glycoprotein G, were rearranged into all six possible orders. Viable viruses were recovered from each of the rearranged cDNAs. The recovered viruses were examined for their levels of gene expression, growth potential in cell culture, and virulence in mice. Gene rearrangement changed the expression levels of the encoded proteins in concordance with their distance from the 3' promoter. Some of the viruses with rearranged genomes replicated as well or slightly better than wild-type virus in cultured cells, while others showed decreased replication. All of the viruses were lethal for mice, although the time to symptoms and death following inoculation varied. These data show that despite the highly conserved gene order of the Mononegavirales, gene rearrangement is not lethal or necessarily even detrimental to the virus. These findings suggest that the conservation of the gene order observed among the Mononegavirales may result from immobilization of the ancestral gene order due to the lack of a mechanism for homologous recombination in this group of viruses. As a consequence, gene rearrangement should be irreversible and provide an approach for constructing viruses with novel phenotypes.

Gene rearrangement attenuates expression and lethality of a nonsegmented negative strand RNA virus.

The nonsegmented negative strand RNA viruses comprise hundreds of human, animal, insect, and plant pathogens. Gene expression of these viruses is controlled by the highly conserved order of genes relative to the single transcriptional promoter. We utilized this regulatory mechanism to alter gene expression levels of vesicular stomatitis virus by rearranging the gene order. This report documents that gene expression levels and the viral phenotype can be manipulated in a predictable manner. Translocation of the promoter-proximal nucleocapsid protein gene N, whose product is required stoichiometrically for genome replication, to successive positions down the genome reduced N mRNA and protein expression in a stepwise manner. The reduction in N gene expression resulted in a stepwise decrease in genomic RNA replication. Translocation of the N gene also attenuated the viruses to increasing extents for replication in cultured cells and for lethality in mice, without compromising their ability to elicit protective immunity. Because monopartite negative strand RNA viruses have not been reported to undergo homologous recombination, gene rearrangement should be irreversible and may provide a rational strategy for developing stably attenuated live vaccines against this type of virus.

Cotranslational disassembly of flock house virus in a cell-free system.

Intact, purified particles of the nodaviruses flock house virus and nodamura virus that were either transfected into cells that were resistant to infection or introduced into in vitro translation systems directed the synthesis of viral proteins. We infer that direct interaction of these nodavirus particles with cytoplasmic components mediated virion disassembly that resulted in release of the viral RNA.

Replication of flock house virus RNAs from primary transcripts made in cells by RNA polymerase II.

To develop vector systems that combine high transcription activity with biologically safe delivery vehicles, we have explored the use of RNA replication to amplify mRNAs, by using flock house virus (FHV) as a model system. The FHV RNA replicase is encoded in the larger of the two segments that comprise the viral positive-sense RNA genome. A cDNA copy of this self-replicating RNA was precisely positioned between a promoter site for cellular RNA polymerase II and a cDNA encoding a self-cleaving ribozyme from hepatitis delta virus. Transfection of this plasmid into cultured BHK cells resulted in prolonged, autonomous FHV RNA replication in the cytoplasm and substantial amplification of the RNA replicon. The replicase also amplified RNA transcribed from a second plasmid of similar design that contained a cDNA copy of the other FHV genome segment. These results constitute a significant step toward the harnessing of nodaviral RNA replication as the basis of a versatile vector system.

Definition and functional analysis of the signal/anchor domain of the human respiratory syncytial virus glycoprotein G.

The attachment protein G of human respiratory syncytial (RS) virus is a type II transmembrane glycoprotein. A secreted from of the G protein is also produced. To examine the two distinct hydrophobic regions in the N-terminal 63 amino acids of G protein for their role(s) in membrane insertion and anchoring, transport to the cell surface, and secretion, G proteins that contained point mutations or deletions were synthesized by cell-free transcription-translation and in cells by expression from recombinant vaccinia virus vectors. A mutant protein lacking the entire major hydrophobic region (amino acids 38-63) was not glycosylated, not expressed on the cell surface, and not secreted, because it was not inserted into membranes. In contrast, deletion of the minor hydrophobic region (amino acids 23-31) had no detectable effect on membrane insertion or anchoring. These data provided direct evidence that amino acids 38-63 were necessary for membrane insertion and contained the signal/anchor domain of RS virus G protein. Mutant proteins that lacked either the N-terminal or the C-terminal half of this 26 residue hydrophobic region were inserted into membranes and processed to maturity, showing that either half of this region was sufficient for membrane insertion. However, these two mutant proteins were secreted more abundantly than wild-type G protein. We propose that their truncated hydrophobic domains interacted with membranes in a way that mimicked the N-terminal signal sequence of naturally secreted proteins, allowing proteolytic cleavage of the mutant proteins.

Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones.

Infectious vesicular stomatitis virus (VSV), the prototypic nonsegmented negative-strand RNA virus, was recovered from a full-length cDNA clone of the viral genome. Bacteriophage T7 RNA polymerase expressed from a recombinant vaccinia virus was used to drive the synthesis of a genome-length positive-sense transcript of VSV from a cDNA clone in baby hamster kidney cells that were simultaneously expressing the VSV nucleocapsid protein, phosphoprotein, and polymerase from separate plasmids. Up to 10(5) infectious virus particles were obtained from transfection of 10(6) cells, as determined by plaque assays. This virus was amplified on passage, neutralized by VSV-specific antiserum, and shown to possess specific nucleotide sequence markers characteristic of the cDNA. This achievement renders the biology of VSV fully accessible to genetic manipulation of the viral genome. In contrast to the success with positive-sense RNA, attempts to recover infectious virus from negative-sense T7 transcripts were uniformly unsuccessful, because T7 RNA polymerase terminated transcription at or near the VSV intergenic junctions.

Fidelity of homologous recombination in vaccinia virus DNA.

The fidelity of homologous recombination in vaccinia virus (VV) DNA was examined by constructing a viral recombinant whose genome contained a copy of the Escherichia coli lac z gene in which the central third of the gene was repeated on either side of the VV thymidine kinase (tk) gene. In this virus, homologous DNA recombination and consequent excision of the tk gene were necessary to restore the open reading frame for beta-galactosidase and thereby to confer a lac z+ phenotype. Imprecise recombination was predicted to increase the frequency of lac z- virus. However, after several passages during which almost every viral genome underwent homologous recombination, the frequency of lac z- plaques was indistinguishable from that of a control virus that could express beta-galactosidase without prior recombination. We conclude that homologous recombination in VV DNA occurs with perfect fidelity at least 99% of the time.