Serial Undiluted Passage Research Articles

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

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.

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.

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.

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.

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.

家蚕核多角体病ウイルスの欠損ゲノムの生成

A plaque-purified Bombyx mori nuclear polyhedrosis virus, BmNPV D1 isolate, was propagated for 30 generations by serial undiluted passages in BmN4 cells. During the passages, a few polyhedra phenotypes and defective viral genomes were generated. By contrast, the population of wild type polyhedra and genomes decreased. The main component of defective genomes contained most of the interspersed homologous sequences of the baculovirus genomes.

Molecular cloning and characterization of a Sendai virus internal deletion defective RNA

A small defective Sendai virus RNA was selectively amplified from a virus preparation obtained after serial undiluted passages in embryonated eggs. Preliminary characterization showed that this defective RNA was a true internal deletion defective RNA, containing the 5' and 3' ends of the non-defective viral genomic RNA. Cloning of this RNA after reverse transcription and polymerase chain reaction amplification was performed in such a way that an exact copy of the defective RNA could be obtained by transcription of the plasmid with T7 RNA polymerase. Sequence analysis of the plasmid allowed further characterization of the defective RNA. It was shown potentially to encode a C-terminally truncated nucleocapsid (NP) protein of 162 amino acids. This truncated NP protein was identified in cells naturally infected with the defective virus preparation. Moreover the protein produced was shown to correspond to the protein synthesized in vitro from the T7 polymerase transcript of the cloned defective genome.

Location of Two Putative Origins of DNA Replication of Autographa californica Nuclear Polyhedrosis Virus

Previously, we described a defective Autographa californica nuclear polyhedrosis virus (AcMNPV) which must contain cis-acting elements required for DNA synthesis, such as the origin(s) of replication (ori). Defective genomes of AcMNPV generated after serial undiluted passage were analyzed further. Three small separated regions were retained in DNA of defective AcMNPV and accumulated in extracellular defective interfering viruses as well as in intracellular DNA after 40 passages. Two of these regions have now been identified as containing putative ori . They are located on the HindIII-B fragment between map units (m.u.) 50.1 and 53.2 and on the HindIII-Q fragment between m.u. 87.2 and 88.9 of the physical map of AcMNPV DNA, respectively. Transfection of Spodoptera frugiperda cells with plasmids containing these sequences followed by superinfection with intact helper AcMNPV resulted in amplification of these plasmids, as demonstrated by the DpnI sensitivity assay. The replicating activity of HindIII-Q is putatively located within the 1000-bp region containing a highly repetitive DNA (hr5), which is also ascribed to enhance delayed-early gene expression. In order to demonstrate replicating activity of test plasmids, it appeared essential to transfect the cells well before superinfection with helper virus.

Lowered Affinity of Structural Protein VP2 to Monoclonal Antibodies in Serial Undiluted Passage of Infectious Pancreatic Necrosis Virus(IPNV).

Lowered Affinity of Structural Protein VP2 to Monoclonal Antibodies in Serial Undiluted Passage of Infectious Pancreatic Necrosis Virus(IPNV).

Detection and analysis of Autographa californica nuclear polyhedrosis virus mutants with defective interfering properties

Defective interfering particles (DIPs) were generated upon continuous production of Autographa californica nuclear polyhedrosis virus (AcNPV) in bioreactors. This configuration mimicked the serial undiluted passaging of virus, which is known to result in plaque-morphology mutants. Restriction enzyme analysis of DIP-containing preparations of extracellular virus showed the presence of many DNA fragments in less than equimolar amounts. These fragments were colinear on the physical map of AcNPV and extended from map position 1.7 to 45. These DIPs thus lacked 43% of the genetic information of the standard virus, including the polyhedrin and DNA polymerase genes. The existence of DIPs was confirmed by electron microscopy, where virions were observed with reduced length. Among the less than equimolar fragments in DIP-containing preparations, fragments were observed linking sequences from map positions 1.7 and 45 via a TGTT linker of unknown origin. The DIPs could not be plaque-purified and needed standard (helper) virus to replicate; DIP-containing preparations interfered with standard virus replication in an interference assay, which explained the reduction in productivity of an AcNPV expression vector-insect cell system in continuous bioreactor operations. The origin of these DIPs and their possible generation mechanism are discussed.

A detailed analysis of duplications appearing during early, high multiplicity infections with polyoma virus.

Serial undiluted passage of polyoma virus derived from transfection of mouse fibroblasts with well defined wild-type genomes results in the appearance of a very heterogeneous population of defective virus particles. Many of these variants show duplications which not only contain the cis-acting region minimally required for efficient replication, but also other regions. A detailed analysis of the duplication patterns appearing in high multiplicity infections is presented. We performed heteroduplex analyses of duplicated fragments using mung bean nuclease and demonstrated that the pattern of duplication junctions is conserved qualitatively and quantitatively. However, the distribution of fragment sizes varied in a number of independently derived virus stocks. Amplification of viral nucleotide sequences is an early event in virus replication, occurring at least as early as 3 days post-transfection. The pattern of duplication did not change significantly in successive early passages at high multiplicity. Although duplication was accompanied by deletion of various parts of the viral genome, a sequence bordering the duplications at the late side of the origin of replication was retained as a single copy in all defective viruses. The relevance of these findings to the mechanism that creates the duplications and the biological activity of defective virus is discussed.

Characterization of defective interfering RNAs of Berne virus

Defective interfering (DI) RNAs of Berne virus (BEV) were generated by serial undiluted passaging of the virus in embryonic mule skin cells. Two DI RNAs of 1.0 and 1.4 kb (designated DI1000 and DI1400) were characterized in more detail. Isokinetic sucrose gradient analysis showed that these DI RNAs are specifically packaged into particles with smaller S values than standard virions. Both DI RNAs were cloned and sequenced. Three genomic cDNA clones were identified using probes complementary to the 5' end of a DI RNA, which are thought to be derived from the 5'-terminal region of the BEV genome. A non-translated region of about 700 nt and the 5' end of the putative BEV replicase gene were identified in the consensus nucleotide sequence. Both DI RNAs were shown to contain sequences from the 5' and 3' ends of the BEV genome. A conserved sequence motif, which has been postulated to be involved in sub-genomic RNA transcription, was also identified just downstream of the extreme 5' ends of DI1000 and DI1400.

Viral Polypeptides and RNA of Defective Interfering Particles of IPN Virus Generated by Serial Undiluted Passaging in TO-2 Cells.

HB-1 virus, a strain of infectious pancreatic necrosis virus (IPNV), was isolated from hard clam, Meretrix lusoria, with TO-2 cell line derived from tilapia ovary. The defective interfering (DI) particles of HB-1 virus could be generated by serial undiluted passaging. The polypeptides and RNA in virus particles of diluted and undiluted virus preparations were compared in order to investigate the nature of defective interfering particles involved in viral interference of HB-1 virus. Cesium chloride density gradient centrifugation was applied to purify virus particles of diluted and undiluted virus preparations. By comparing the polypeptides and RNA in virus particles of diluted and undiluted virus preparations, it was revealed that (I) the virus particles of undiluted virus preparation had smaller polypeptides than normal ; (II) subgenomic RNA of large segment was found in virus particles of undiluted virus preparation. These two findings suggested that DI particles of HB-1 virus was a deletion mutant of standard virus.This is the first report indicating the polypeptides and RNA possibly related to IPN virus DI particles.