Undiluted Passage Research Articles

My Pathway to Adeno-Associated Virus and Adeno-Associated Virus Gene Therapy: A Personal Perspective.

My Pathway to Adeno-Associated Virus and Adeno-Associated Virus Gene Therapy: A Personal Perspective.

Integrative effect of defective interfering RNA accumulation and helper virus attenuation is responsible for the persistent infection of Japanese encephalitis virus in BHK‐21 cells

Persistence of RNA viruses is often, but not always, associated with the production of defective interfering (DI) particles. To investigate possible roles of DI particles and helper viruses in RNA virus persistence, persistent infection with Japanese encephalitis virus (JEV) was established in baby hamster kidney (BHK-21) cells. At the 6th and 7th serial undiluted passages of JEV on BHK-21 cells, viral persistence was established spontaneously with DI RNA generation. Seven cell clones exhibiting persistent infection were obtained from the initial BHK-21 cell batches exhibiting JEV persistence, and maintained for over 400 days. Most cell clones produced infectious particles (10(1) -10(5) PFU/ml) continuously, expressed viral proteins, and resisted homologous superinfection. Two helper viruses, chvBS6-3 and chvBS7-1, were isolated from two of the seven cell clones, and characterized to investigate their roles in JEV persistence. While chvBS6-3 was restored to its full cytopathicity in the absence of DI RNA, chvBS7-1 exhibited almost no cytopathicity, regardless of DI RNA co-replication. Attenuation of chvBS7-1 did not appear to be due to inadequate adsorption or genome replication, but due to inefficient egress of the assembled progeny virions, suggesting altered helper virus emergence during JEV persistence in BHK-21 cells. These observations suggest that at least two mechanisms are involved in JEV persistence; a DI RNA-dependent mechanism, where DI RNA co-replication nullifies the helper virus's cytopathicity, or a DI RNA-independent mechanism, where the helper virus is self-attenuated. This study provides a useful in vitro tool for understanding the mechanisms underlying RNA virus persistent infections.

Interference of BmNPV replication by the viruses altered by serial undiluted passage in cell culture

Interference of BmNPV replication by the viruses altered by serial undiluted passage in cell culture

Establishment of an insect cell clone that harbours a partial baculoviral genome and is resistant to homologous virus infection

After serially undiluted passage of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV), persistently infected Se301 cells were established. A cell strain, in which no polyhedra or viral particles were observed, was cloned and designated P8-Se301-C1. The P8-Se301-C1 cells are morphologically similar to but grow slower than Se301 cells and they can homologously interfere with SeMNPV. PCR analysis showed that SeMNPV ie-0 and polyhedrin genes were present but DNA polymerase and orf67 genes were absent in P8-Se301-C1, suggesting that the cells harbour incomplete SeMNPV genomes. Dot-blot analysis demonstrated that 0.32+/-0.16 ng SeMNPV DNA was present in 1.25 x 10(5) P8-Se301-C1 cells. A quantitative real-time PCR assay showed that there were 13.2+/-4.3 copies of the SeMNPV polyhedrin gene in each cell. Nested RT-PCR demonstrated the presence of SeMNPV polyhedrin transcripts in P8-Se301-C1 cells. The fact that P8-Se301-C1 cells carry low levels of partial viral genome but do not produce viral progeny suggests a latent-like viral infection in the cells.

The pleiotropic cost of host-specialization in Tobacco etch potyvirus

Host-range expansion is thought to allow viruses to broaden their ecological niches by allowing access to new resources. However, traits governing the infection of multiple hosts may decrease fitness in the original one due to the pleiotropic effect of adaptive mutations that may give rise to fitness tradeoffs across hosts. Here, we have experimentally examined the consequences of host-specialization in the plant pathogen Tobacco etch potyvirus (TEV). Replicate populations of TEV were allowed to evolve for 15 serial undiluted passages on the original tobacco host or on pepper, a novel host. Virulence and biologically active viral load were evaluated during the course of the experiment for each lineage on both potential hosts. In agreement with the tradeoff hypothesis, lineages evolved in the novel host experienced substantial increases in virulence and virus accumulation in its own host, but suffered reduced virulence and accumulation on the original host. By contrast, lineages evolved on the ancestral host did not increase virulence or viral load on either host. Genomic consensus sequences were obtained for each lineage at the end time point. The potential relevance for the evolution of virulence and virus fitness of the characterized mutations is discussed.

Porcine Rotavirus Bearing an Aberrant Gene Stemming from an Intergenic Recombination of the NSP2 and NSP5 Genes Is Defective and Interfering

Serial undiluted passage of a porcine rotavirus in MA104 cells yielded three distinct virus populations, each of which bore different rearranged genes. Sequencing revealed that each of two populations bore a distinct intragenic recombinant NSP3 gene consisting of a partial duplication in a head-to-tail orientation without altering the NSP3 open reading frame and the third population carried both an intragenic recombinant NSP3 gene and an intergenic recombinant gene (1,647 nucleotides in length) which contained a truncated NSP2 gene inserted into the NSP5 gene at residue 332. The former two populations were viable, whereas the latter population was defective and interfering.

Sequences conserved in the defective interfering RNAs of Semliki Forest virus: an electron microscopic heteroduplex analysis.

Alanen, M., Wartiovaara, J. and Soderlund, H. 1987. Sequences conserved in the defective interfering RNAs of Semliki Forest Virus: An electron microscopic heteroduplex analvsis. - Hereditas 106: 19–29. Lund, Sweden. ISSN 00184661. The RNA structure of three species of defective interfering particles (DI) that have been generated by undiluted passaging of Semliki Forest virus was determined by heteroduplex analysis of Complementary DNA. Hybridization of the three cDNAs with two previously sequenced DI cDNA species leads to the elucidation of the sequences common to the five different DIs. There are altogether about 270 nucleotides in three separate sequence blocks (80, 90 and 100 nucleotides) that have been conserved through passaging. Signals for the process of replication and encapsidation must have been retained in these sequences. One of the sequences contains a 20 nucleotides long region with a palindromic sequence also found in the DI-RNAs of the closely related Sindbis virus.

Rubella virus DI RNAs and replicons: requirement for nonstructural proteins acting in cis for amplification by helper virus.

A rubella virus (RUB) replicon was constructed by replacing the 3' proximal structural protein ORF (SP-ORF) in Robo402, a RUB infectious cDNA clone, with a reporter gene, green fluorescent protein (GFP). This replicon, RUBrep/GFP, mimics naturally occurring RUB defective-interfering (DI) RNAs generated during serial undiluted passage that maintain the 5' proximal nonstructural protein ORF (NS-ORF) but contain deletions in the SP-ORF. Following transfection of Vero cells with in vitro RNA transcripts from RUBrep/GFP, replicon replication occurred and the replicon was amplified and spread to other cells in the presence of standard helper virus. GFP expression was a much more sensitive indicator of replicon replication than was Northern analysis to detect replicon-specific RNAs. Most of a series of RUBrep/GFP constructs with deletions in the NS-ORF not only were incapable of self-replication, but were not amplified by standard helper virus. The only exception was a construct with an in-frame deletion between two NotI sites that removed nucleotides 1685-2192 of the genome; this construct did not express GFP by itself, but did express GFP in the presence of standard helper RUB and was spread to other cells. Thus, with the exception of this region, the NS-ORF is required in cis for amplification of RUB replicons by standard helper virus, explaining the selection of DI RNAs that maintain the NS-ORF. Surprisingly, when the NotI deletion was introduced into Robo402, a viable virus resulted that replicated only threefold less efficiently than did Robo402 virus. Thus, the NotI region of the NS-ORF is not necessary for virus replication. This deletion covers a region of the NS-ORF without predicted function, which therefore may function as a spacer or hinge between functional domains. Nevertheless, it was an unexpected finding that a small virus such as RUB could dispense with approximately 10% of its genome.

Isolation and characterization of an arterivirus defective interfering RNA genome.

Equine arteritis virus (EAV), the type member of the family Arteriviridae, is a single-stranded RNA virus with a positive-stranded genome of approximately 13 kb. EAV uses a discontinuous transcription mechanism to produce a nested set of six subgenomic mRNAs from which its structural genes are expressed. We have generated the first documented arterivirus defective interfering (DI) RNAs by serial undiluted passaging of a wild-type EAV stock in BHK-21 cells. A cDNA copy of the smallest DI RNA (5.6 kb) was cloned. Upon transfection into EAV-infected BHK-21 cells, transcripts derived from this clone (pEDI) were replicated and packaged. Sequencing of pEDI revealed that the DI RNA was composed of three segments of the EAV genome (nucleotides 1 to 1057, 1388 to 1684, and 8530 to 12704) which were fused in frame with respect to the replicase reading frame. Remarkably, this DI RNA has retained all of the sequences encoding the structural proteins. By insertion of the chloramphenicol acetyltransferase reporter gene in the DI RNA genome, we were able to delimitate the sequences required for replication/DI-based transcription and packaging of EAV DI RNAs and to reduce the maximal size of a replication-competent EAV DI RNA to approximately 3 kb.

Ebola Virus Defective Interfering Particles and Persistent Infection

Ebola virus (Zaire subtype) is associated with high mortality disease outbreaks that commonly involve human to human transmission. Surviving patients can show evidence of prolonged virus persistence. The potential for Ebola virus to generate defective interfering (DI) particles and establish persistent infections in tissue culture was investigated. It was found that serial undiluted virus passages quickly resulted in production of an evolving population of virus minireplicons possessing both deletion and copyback type DI genome rearrangements. The tenth undiluted virus passage resulted in the establishment of virus persistently infected cell lines. Following one or two crises, these cells were stably maintained for several months with continuous shedding of infectious virus. An analysis of the estimated genome lengths of a selected set of the Ebola virus minireplicons and standard filoviruses revealed no obvious genome length rule, such as “the rule of six” found for the phylogenetically related Paramyxovirinae subfamily viruses. Minimal promoters for Ebola virus replication were found to be contained within 156 and 177 nucleotide regions of the genomic and antigenomic RNA 3′ termini, respectively, based on the length of authentic termini retained in the naturally occurring minireplicons analyzed. In addition, using UV-irradiated preparations of virus released from persistently infected cells, it was demonstrated that Ebola virus DI particles could potentially be used as natural minireplicons to assay standard virus support functions.

Genome Nucleotide Lengths That Are Divisible by Six Are Not Essential but Enhance Replication of Defective Interfering RNAs of the Paramyxovirus Simian Virus 5

For some members of the Paramyxoviridae family of negative strand RNA viruses, efficient genome replication only occurs when the total genome length is a multiple of six (6Nlength, whereNis any integer). To determine if this “rule of six” requirement applied to the replication of the prototype paramyxovirus simian virus 5 (SV5), defective interfering (DI) RNA genomes were generated by sequential undiluted passage of virus in tissue culture. Molecular cloning and nucleotide sequence analysis of 10 RNA genomes revealed a series of copyback DI RNAs with chain lengths between 449 and 1365 bases, but only 4 of the 10 naturally occurring RNA genomes were of 6Nlength. Many of the cloned DI genomes could be grouped into two distinct nested sets, with the members of each set having the same polymerase crossover junctions and extent of terminal complementarity but differing from each other by internal deletions. One of these nested sets of genomes consisted of novel DI RNAs that contained a pentameric stretch of nontemplated adenosine residues inserted precisely at the polymerase crossover junction. A reverse genetics system was established in which SV5 DI genomes were replicatedin vivoentirely by cDNA-derived components. Using this system, two naturally occurring SV5 DI RNAs were examined in a mutational analysis to determine the role of genome length on SV5 RNA replication. The progressive insertion of one to six nucleotides into a 6Nlength DI genome (852 bases) resulted in a reduction in replication for RNAs that contained one to four additional bases (∼35–50% of WT levels), followed by an increase back to WT replication levels for genomes that were altered by five and six base insertions (∼70 and 100% of WT levels, respectively). An insertion of five nucleotides into a second non-6Nlength DI RNA (499 total bases) created a genome length that was a multiple of six (504 bases) and led to a ∼10-fold stimulation of replication over that of the unaltered genome. Together, these results indicate that there was a clear influence of 6Ngenome length on SV5 DI RNA replication, but the stringency of this replication requirement appeared to be less than that found previously for other paramyxoviruses. This work completes the testing of the rule of six replication requirement for representatives of each of the four genera of the Paramyxoviridae family and indicates that the preference for replication of 6Nlength RNA genomes varies between the individual paramyxoviruses.

Sindbis virus replicons and Sindbis virus: assembly of chimeras and of particles deficient in virus RNA.

Alphaviruses are a well-characterized group of positive-strand RNA viruses. The identification of cis-acting elements in their genomes and their replication strategy have made them useful as vectors for the expression of heterologous genes. In infected cells, the nonstructural proteins, required for replication and transcription of the viral genes, are translated from the genomic RNA; the structural proteins, the capsid protein that interacts with the RNA to form the nucleocapsid and the proteins embedded in the lipid envelope, are translated from a subgenomic mRNA and can be replaced by heterologous genes. Such modified genomes are self-replicating (replicons); they can be introduced into the cells by transfection and can also be packaged into extracellular particles with defective helper (DH) RNAs. The particular DH RNA determines how well it is replicated and to what extent it is packaged. One potential complication of this system has been that recombination between the replicon genome and the DH RNA may occur. The studies described here were designed to prevent recombination by expressing the capsid protein from one DH RNA and the virus membrane proteins from a second helper RNA. Recombination to yield a nonsegmented infectious virus genome would then require several independent crossover events. There is a translational enhancer located downstream of the initiating AUG in the RNA of the capsid gene that had to be conserved in the second helper to achieve high-level expression of the viral glycoproteins. For this reason, we modified the capsid protein gene in two ways: the first was to use the capsid protein gene from a different alphavirus, Ross River virus, and the second was to make deletions in that gene to maintain the translational enhancer in the RNA but to eliminate the positively charged region in the protein that should be essential for the specific and nonspecific interactions with RNA. Transfections with replicon RNA and the deleted chimeric DH RNA as the only helper resulted in the high-level production of particles that were almost completely devoid of RNA. The inclusion of a helper expressing an intact Sindbis virus capsid protein gene led to the production of high levels of packaged replicons. Recombinants were not detected even after several undiluted passages.

Long-Term, Large-Population Passage of Aphthovirus Can Generate and Amplify Defective Noninterfering Particles Deleted in the Leader Protease Gene

During serial undiluted passage of a clonal population of foot-and-mouth disease virus (FMDV C-S8c1) in BHK-21 cells, two species of defective RNA were generated and selected. Sequence analysis revealed that they included deletions within the L-coding region, and retained the correct reading frame for viral protein synthesis. These deleted RNAs directed the synthesis of capsid protein VP1, were packaged in particles sedimenting with standard virus, required homologous infectious helper virus in order to produce viral particles, but did not interfere with the replication of helper virus. While detection of defective particles in FMDV required more than 100 serial passages, once produced, these defective RNAs could be stably maintained upon further passages in the FMDV C-S8c1 quasispecies. Furthermore, a high fitness, monoclonal-antibody-resistant virus was able to replace the standard virus and support the amplification of the deleted particles. This is the first description of naturally occurring, defective particles of FMDV.

The Production of Recombinant Infectious DI-Particles of a Murine Coronavirus in the Absence of Helper Virus

We have studied the production and release of infectious DI-particles in vaccinia-T7-polymerase recombinant virus-infected L cells that were transfected with five different plasmids expressing the synthetic DI RNA MIDI-HD and the four structural proteins (M, N, S, and E) of the murine coronavirus MHV-A59. The DI cDNA contains the hepatitis delta ribozyme sequences to generate in the transfected cells a defined 3′ end. In EM studies of transfected cells virus-like particles (VLP) were observed in vesicles. Release of the particles into the medium was studied by immunoprecipitations of proteins released into the culture supernatant. Particle release was independent of S or N, but required M and E. Coexpression of E and M was sufficient for particle release. Coexpression of the structural proteins and the MIDI-HD RNA resulted in the production and release of infectious DI-particles. Infectivity of the DI-particles was determined by adding helper virus MHV-A59 to the medium containing the VLPs and using this mixture to infect new L cells. Intracellular RNA of several subsequent undiluted passages was isolated to detect the MIDI-HD RNA. Passage of the MIDI-HD RNA was dependent on the expression of the structural proteins of MHV-A59 in the transfected cells. In the absence of either E or M, MIDI-HD RNA could not be passaged to fresh L cells. We have thus developed a system in which we can produce coronavirus-like particles and an assay to test their infectivity.

Molecular Characterization of Transmissible Gastroenteritis Coronavirus Defective Interfering Genomes: Packaging and Heterogeneity

Three transmissible gastroenteritis virus (TGEV) defective RNAs were selected by serial undiluted passage of the PUR46 strain in ST cells. These RNAs of 22, 10.6, and 9.7 kb (DI-A, DI-B, and DI-C, respectively) were detected at passage 30, remained stable upon further passage in cell culture, and significantly interfered with helper mRNA synthesis. RNA analysis from purified virions showed that the three defective RNAs were efficiently packaged. Virions of different densities containing either full-length or defective RNAs were sorted in sucrose gradients, indicating that defective and full-length genomes were independently encapsidated. DI-B and DI-C RNAs were amplified by the reverse transcription-polymerase chain reaction, cloned, and sequenced. DI-B and DI-C genomes are formed by three and four discontinuous regions of the wild-type genome, respectively. DI-C contains 2144 nucleotides (nt) from the 5′-end of the genome, two fragments of 4540 and 2531 nt mostly from gene 1b, and 493 nt from the 3′ end of the genome. DI-B and DI-C RNAs include sequences with the pseudoknot motif and encoding the polymerase, metal ion binding, and helicase motifs. DI-B RNA has a structure closely related to DI-C RNA with two main differences: it maintains the entire ORF 1b and shows heterogeneity in the size of the 3′ end deletion. This heterogeneity maps at the beginning of the S gene, where other natural TGEV recombination events have been observed, suggesting that either a process of template switching occurs with high frequency at this point or that the derived genomes have a selective advantage.

The sequential changes of the virion polypeptides of CV HB1 virus (Birnaviridae) during serial undiluted passaging

Clam virus (CV HB-1) isolated from hard clam Meretrix lusoria, typed as infectious pancreatic necrosis virus (IPNV) Ab serotype, was employed for the present study. The structural polypeptides of purified virus generated by serial undiluted and diluted passaging were analyzed using sodiumdodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results indicated that in VP2, the virus particles of serial undiluted passaging had viral polypeptides smaller than those of serial diluted passaging. Furthermore, a polypeptide with a molecular mass of 105 kDa was found in the serial undiluted virus preparation. Further analysis by monoclonal antibodies showed that VP105 might be a polyprotein produced by genomic RNA segment A. To investigate the sequential changes of viral polypeptides of the virus preparation at high multiplicity infection, the viral polypeptides at each undiluted passage were analyzed by SDS-PAGE followed by immunoblotting. The results indicated that VP105 was present by undiluted passage 7, while the smaller VP2 was present by undiluted passage 10. The amount of smaller VP2 and VP105 increased as the number of passages increased.

Structure and encapsidation of transmissible gastroenteritis coronavirus (TGEV) defective interfering genomes.

Serial undiluted passages were performed with the PUR46 strain of TGEV in swine testis (ST) cells. Total cellular RNA was analyzed at different passages after orthophosphate metabolic labeling. Three new defective RNA species of 24, 10.5, and 9.5 kb (DI-A, DI-B, and DI-C respectively) were detected at passage 30, which were highly stable and significantly interfered with helper mRNA synthesis in subsequent passages. By Northern hybridization DIs A, B, and C were detected in purified virions at amounts similar to those of helper RNA. Standard and defective TGEV virions could be sorted in sucrose gradients, indicating that defective and full-length genomes are independently packaged. cDNA synthesis of DI-B and DI-C RNAs was performed by the reverse transcription-polymerase chain reaction (RT-PCR) to give four fragments in each case. Cloning and sequencing of the DI-C PCR products showed that the smallest DI particle comprises 9.5 kb and has 4 discontinuous regions of the genome. It contains 2.1 kb from the 5'-end of the genome, about 7 kb from gene 1b, the first 24 nucleotides of the S gene, 12 nucleotides of ORF 7, and the 0.4 kb of the UTR at the 3'-end.

Characterization of defective-interfering RNAs of rubella virusgenerated during serial undiluted passage

During serial undiluted passage of rubella virus (RUB) in Vero cells, two species of defective-interfering (DI) RNAs of approximately 7000 and 800 nucleotides (nts) in length were generated (Frey, T. K., and Hemphill, M. L., Virology 164, 22–29, 1988). In this study, these DI RNAs were characterized by molecular cloning, hybridization with probes of defined sequence, and primer extension. The 7000-nt DI RNA species were found to be authentic DI RNAs which contain a single 2500- to 2700-nt deletion in the structural protein open reading frame (ORF) region of the genome. The 800-nt RNAs were found to be subgenomic DI RNAs synthesized from the large DI RNA templates. Analysis of the extent of the deletions using a reverse-transcription-PCR protocol revealed that the 3′ end of the deletions did not extend beyond the 3′ terminal 244 nts of the genome. The 5′ end of the deletions did not extend into the nonstructural protein ORF; however, DI RNAs in which the subgenomic start site was deleted were present. Following serial undiluted passage of seven independent stocks of RUB, this was the only pattern of DI RNAs generated. DI RNAs of 2000 to 3000 nt in length were the majority DI RNA species in a persistently infected line of Vero cells, showing that other types of RUB DI RNAs can be generated and selected. However, when supernatant from the persistently infected cells was passaged, the only DI RNAs present after two passages were 7000 nts in length, indicating that this species has a selective advantage over other types of DI RNAs during serial passage.

Co-operation and Defection: Playing the Field in Virus Dynamics

A previous model (Szathmáry, 1992) is further developed for the dynamics of standard (V) and defective interfering (DI) viruses. The crucial retained element is the incorporation of population structure in the form of a complete distribution of cells infected by particles differing in number. New elements are: the non-linear shared benefit from the contribution of Vs to the group of viruses infecting the same cell (synergistic at low numbers, diminishing returns at high numbers, respectively); a dynamics for the total number of particles (V and DI); and the possibility of extinction if the frequency of Vs is small enough. In evolutionary genetical terms this is a frequency- and density-dependent evolutionary game. A crucial result is retained: coexistence of Vs and DIs is possible provided the multiplicity of infection (hence the size of the coinfection group) is large enough. Phase portraits and vector-field plots for a continuous-time and numerical solutions for a corresponding discrete-time case are presented. The latter reflect the basic features of serial, undiluted passage. The causes for two possible means of extinction (low initial frequency of Vs, and very high vigour of Vs) are revealed: the appearance of high amplitude fluctuations. Coexistence can apparently be ensured by stable points, periodic behaviour, or strange attractors. The paper clarifies the dynamical background of cycles found experimentally in V-DI systems.

Natural Evolution of Coronavirus Defective-Interfering RNA Involves RNA Recombination

Defective-interfering (DI) RNAs of RNA viruses, in general, are generated and continue to evolve in size during serial undiluted passages of viruses. This evolution was thought to occur by independent generation of DI RNAs during virus passages and subsequent selection of new DI RNAs under new cellular conditions. Here we demonstrate that recombination between the old DI RNA and the helper viral RNA can be one of the mechanisms for natural DI RNA evolution. A mouse hepatitis virus (MHV) DI RNA, DIssE RNA, was transcribed in vitro and transfected into a mouse cell line infected with a different MHV strain (A59), which is distinguishable from the original natural helper MHV (JHM). During subsequent serial undiluted passages of the harvested virus, several novel DI RNA species were generated, while the original DIssE RNA disappeared by passage 11. cDNA cloning and sequence analysis of one of these novel DI RNAs, designated DI-2, revealed that it is composed of four discontinuous regions of the genomic sequence and is different from the structure of the original DIssE RNA. Sequence comparison among DI-2, DIssE, and helper MHV-A59 RNAs showed that DI-2 sequence is similar to DIssE in the first and second regions, but similar to the helper A59 virus in the third and last regions. Thus, this DI RNA was generated by RNA recombination between the original DIssE RNA and the herper viral RNA. These results indicate that recombination between DI RNA and helper virus RNA can be involved in the natural evolution of DI RNAs.