Replication Of Standard Virus Research Articles

Cell culture-based production and in vivo characterization of purely clonal defective interfering influenza virus particles

BackgroundInfections with influenza A virus (IAV) cause high morbidity and mortality in humans. Additional to vaccination, antiviral drugs are a treatment option. Besides FDA-approved drugs such as oseltamivir or zanamivir, virus-derived defective interfering (DI) particles (DIPs) are considered promising new agents. IAV DIPs typically contain a large internal deletion in one of their eight genomic viral RNA (vRNA) segments. Consequently, DIPs miss the genetic information necessary for replication and can usually only propagate by co-infection with infectious standard virus (STV), compensating for their defect. In such a co-infection scenario, DIPs interfere with and suppress STV replication, which constitutes their antiviral potential.ResultsIn the present study, we generated a genetically engineered MDCK suspension cell line for production of a purely clonal DIP preparation that has a large deletion in its segment 1 (DI244) and is not contaminated with infectious STV as egg-derived material. First, the impact of the multiplicity of DIP (MODIP) per cell on DI244 yield was investigated in batch cultivations in shake flasks. Here, the highest interfering efficacy was observed for material produced at a MODIP of 1E−2 using an in vitro interference assay. Results of RT-PCR suggested that DI244 material produced was hardly contaminated with other defective particles. Next, the process was successfully transferred to a stirred tank bioreactor (500 mL working volume) with a yield of 6.0E+8 PFU/mL determined in genetically modified adherent MDCK cells. The produced material was purified and concentrated about 40-fold by membrane-based steric exclusion chromatography (SXC). The DI244 yield was 92.3% with a host cell DNA clearance of 97.1% (99.95% with nuclease digestion prior to SXC) and a total protein reduction of 97.2%. Finally, the DIP material was tested in animal experiments in D2(B6).A2G-Mx1r/r mice. Mice infected with a lethal dose of IAV and treated with DIP material showed a reduced body weight loss and all animals survived.ConclusionIn summary, experiments not only demonstrated that purely clonal influenza virus DIP preparations can be obtained with high titers from animal cell cultures but confirmed the potential of cell culture-derived DIPs as an antiviral agent.

The Impact of Defective Viruses on Infection and Immunity.

Defective viral genomes (DVGs) are generated during viral replication and are unable to carry out a full replication cycle unless coinfected with a full-length virus. DVGs are produced by many viruses, and their presence correlates with alterations in infection outcomes. Historically, DVGs were studied for their ability to interfere with standard virus replication as well as for their association with viral persistence. More recently, a critical role for DVGs in inducing the innate immune response during infection was appreciated. Here we review the role of DVGs of RNA viruses in shaping outcomes of experimental as well as natural infections and explore the mechanisms by which DVGs impact infection outcome.

Defective (Interfering) Viral Genomes Re-Explored: Impact on Antiviral Immunity and Virus Persistence

Defective viral genomes (DVGs) are natural products of virus replication that occur in many positive and negative sense RNA viruses, including Ebola, dengue and respiratory syncytial virus. DVGs, which have severe genomic truncations and require a helper virus to replicate, have three well-described functions: interference with standard virus replication, immunostimulation, and establishment of virus persistence. These functions of DVGs were first described almost 50 years ago, yet only recent studies have shown the molecular intersection between their immunostimulatory and pro-persistence activities. Here, we review more than half a century of scientific literature on the immunostimulatory and pro-persistence functions of DVGs. We highlight recent advances in the field and the critical role DVGs have in both the acute and long-term virus–host interactions.

The UL4 protein of equine herpesvirus 1 is not essential for replication or pathogenesis and inhibits gene expression controlled by viral and heterologous promoters

Defective interfering particles (DIP) of equine herpesvirus 1 (EHV-1) inhibit standard virus replication and mediate persistent infection. The DIP genome is comprised of only three genes: UL3, UL4, and a hybrid gene composed of portions of the IR4 ( EICP22) and UL5 ( EICP27) genes. The hybrid gene is important for DIP interference, but the function(s) of the UL3 and UL4 genes are unknown. Here, we show that UL4 is an early gene activated solely by the immediate early protein. The UL4 protein (UL4P) was detected at 4 hours post-infection, was localized throughout the nucleus and cytoplasm, and was not present in purified virions. EHV-1 lacking UL4P expression was infectious and displayed cell tropism and pathogenic properties in the mouse model similar to those of parental and revertant viruses. Reporter assays demonstrated that the UL4P has a broad inhibitory function, suggesting a potential role in establishing and/or maintaining DIP-mediated persistent infection.

Biological and genotypic properties of defective interfering particles of equine herpesvirus 1 that mediate persistent infection

Infection with equine herpesvirus 1 (EHV-1) preparations enriched for defective interfering particles (DIP) leads to a state of persistent infection in which infected cells become lysis resistant and release both infectious (standard) virus and DIP. EHV-1 DIP are unique in that the recombination events that generate DIP genomes produce new open reading frames (ORFs; Hyb1.0 and Hyb2.0) consisting of 5′ sequences of varying lengths of the early regulatory gene IR4 fused to 3′ sequences of varying lengths of the UL5 regulatory gene. Only two additional ORFs ( UL3 and UL4) are conserved. Because persistently infected cells release a heterogeneous mixture of DIP, characterization of the elements responsible for this altered state of infection has proved difficult. Here we describe a method for studying persistent infection using recombinant DIP (rDIP). Infection with rDIP resulted in the production of recombinant DIP that replicated faithfully to, at least, five passages and mediated a rapid progression to persistent infection as measured by: 1) production of cells resistant to lysis by the standard virus; and 2) infected cells that released both standard virus and DIP. High concentrations of rDIP also resulted in interference with the standard virus replication, another hallmark of persistent infection. rDIP deleted of UL3, UL4, and either Hyb gene, the only functional genes conserved in the DIP genome, replicated but exhibited markedly reduced ability to interfere with standard virus replication. Restoring only the Hyb genes (either Hyb1.0 or Hyb2.0), the IR4 gene, or specific portions of the IR4 gene restored interference. These data suggest that residues 144 to 196 of the IR4 protein within the HYB proteins are important for DIP interference and that persistent infection results from recombination events that produce DIP genomes.

Characterization of Toscana Virus-Defective Interfering Particles Generatedin Vivo

Toscana (TOS) virus stocks strongly interfering with standard virus replication were obtained by sequential passages of virus in suckling mouse brain. Characterization of viral RNAs in these stocks showed the presence of a heterogeneous population of defective RNA molecules derived from the L genomic segment, in both nucleocapsid (NC) and messenger RNAs, suggesting that these molecules could be replicated, assembled, and transcribed. Subgenomic RNAs from the L segment but not from the S or M segments were found in cells infected with these stocks. Defective RNA molecules interfered with virus replication and retained 5′ and 3′ genomic termini. Nucleotide sequence analysis of some cloned defective interfering (DI) RNAs revealed they contained one or more internal deletions reducing their length to 7–13% of the full-length L segment. An identical sequence motif, of variable length, was found at both terminal sites of the RNA junction on standard L sequences. This motif was retained only in one copy in the subgenomic RNA. These results are consistent with the generation of TOS virus DI particlesin vivoand suggest that the defective genomic RNAs could be generated by polymerase jumping from a sequence to an identical one spatially closed because of the RNA structure.

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.

Molecular basis for interference of defective interfering particles of pseudorabies virus with replication of standard virus.

Serial passage of pseudorabies virus (PrV) at high multiplicity yields defective interfering particles (DIPs), but the sharp cyclical increases and decreases in titer of infectious virus that are observed upon continued passage at high multiplicity of most DIPs of other viruses are not observed with DIPs of PrV (T. Ben-Porat and A. S. Kaplan, Virology 72:471-479). We have studied the dynamics of the interactions of the virions present in a population of DIPs to assess the cis functions for which the genomes of the DIPs are enriched. The defective genomes present in one population of DIPs, [PrV(1)42], replicate preferentially over the nondefective genomes present in that virion population at early stages of infection, indicating that the DIP DNA is enriched for sequences that can serve as origins of replication at early stages of infection. This replicative advantage of the DIP DNA is transient and disappears at later stages of infection. The defective DNA does not appear to be encapsidated preferentially over the nondefective DNA present in this virion population, which might indicate that it is not enriched for cleavage-encapsidation sites. However, the nondefective DNA in the DIP virion population has become modified and has acquired reiterations of sequences originating from the end of the unique long (UL) region of the genome. Furthermore, both the infectious and defective genomes present in the DIP population compete for encapsidation more effectively than do the genomes of standard PrV. These results indicate that the defective genomes in the population of virions studied are enriched not only for an origin of replication but probably also for sequences necessary for efficient cleavage-encapsidation. Furthermore, the nondefective genomes present in this population of DIPs have also been modified and have acquired the ability to compete with the defective genomes for cleavage-encapsidation.

Properties of defective interfering particles of Sindbis virus generated in vertebrate and mosquito cells.

Defective interfering (DI) particles of Sindbis virus were generated during serial, undiluted passage of a cloned virus stock in both chick embryo fibroblast (CEF) cells and Aedes albopictus mosquito cells. DI particle-containing stocks were identified by their ability to interfere with standard virus replication and to synthesize small DI RNA species in the cell type in which they were generated. DI RNA species generated during serial passage in CEF cells were able to replicate in both vertebrate and A. albopictus cells. Some of the DI RNA species generated during serial passage in A. albopictus cells replicated efficiently in both cell types while others, sometimes present in the same DI particle stock, failed to replicate in cells of vertebrate origin. These results suggest that evolution of DI RNA species during serial passage of Sindbis virus in mosquito cells may result in the loss or alteration of nucleotide sequences required for RNA synthesis in vertebrate cells.

A replication-efficient mutant of West Nile virus is insensitive to DI particle interference

A previous report described the isolation of a mutant of West Nile virus (WNV) from culture fluid obtained from persistently infected genetically resistant C3H/RV mouse cells that replicates significantly more efficiently in cultures of C3H/RV cells than does the parental virus. This replication-efficient mutant, designated RE-WNV, has now been found to be insensitive to interference by WNV defective interfering (DI) particles. This characteristic was demonstrated by several means. The RE-WNV mutant was able to superinfect persistently infected cultures that were no longer producing detectable parental virus, while the parental virus was not. Good yields of the mutant virus were produced during six serial undiluted passages of RE-WNV in both resistant C3H/RV and congenic susceptible C3H/HE cells. In contrast, during passage of parental virus in C3H/RV cells, progeny virus could not be detected after the third passage, due to an enhanced interference by WNV DI particles with standard virus replication in these cells. The RE-WNV was also insensitive to interference by a pool of parental virus enriched for DI particles. Analysis of the mutant genome by oligonucleotide fingerprinting indicated that the genome RNA of the mutant differs by two unique spots from the parental RNA. The relevance of this mutant to the eventual understanding of the mechanism by which C3H/RV and C3H/HE cells manifest their flavivirus-specific difference in the efficiency of progeny virus production is discussed.

Analysis of extracellular West Nile virus particles produced by cell cultures from genetically resistant and susceptible mice indicates enhanced amplification of defective interfering particles by resistant cultures

[3H]uridine-labeled extracellular West Nile virus (WNV) particles produced by cell cultures obtained from genetically resistant C3H/RV and congenic susceptible C3H/HE mice were compared by sucrose density gradient centrifugation as well as by analysis of the particle RNA. Defective interfering (DI) WNV particles were observed among progeny produced during acute infections in both C3H/RV and C3H/HE cells. Although only a partial separation of standard and DI particles was achieved, the DI particles were found to be more dense than the standard virions. Particles containing several species of small RNAs consistently constituted a major proportion of the total population of virus progeny produced by C3H/RV cells, but a minor proportion of the population produced by C3H/HE cells. Decreasing the multiplicity of infection or extensive plaque purification of the WNV inoculum decreased the proportion of small RNAs found in the progeny virus. The ratio of DI particles to standard virus observed in progeny virus was determined by the cell type used to grow the virus. The ratio could be shifted by passaging virus from one cell type to the other. Homologous interference could be demonstrated with WNV produced by C3H/RV cells but not with virus produced by C3H/HE cells. Continued passage of WNV in C3H/HE cells resulted in a cycling of infectivity. However, passage in C3H/RV cells resulted in the complete loss of infectious virus. Four size classes of small viral RNA, with sedimentation coefficients of about 8, 15, 26, and 34S, were observed in the extracellular particles. A preliminary analysis of these RNAs by oligonucleotide fingerprinting indicated that the smaller RNAs were less complex than the 40S RNA and differed from each other. The data are consistent with the conclusion that WNV DI particles interfere more effectively with standard virus replication and are amplified more efficiently in C3H/RV cells than in congenic C3H/HE cells. The relevance of these findings to the further understanding of genetically controlled resistance to flaviviruses is discussed.

Structure and expression of class II defective herpes simplex virus genomes encoding infected cell polypeptide number 8.

Defective genomes present in serially passaged virus stocks derived from the tsLB2 mutant of herpes simplex virus type 1 were found to consist of repeat units in which sequences from the U(L) region, within map coordinates 0.356 and 0.429 of standard herpes simplex virus DNA, were covalently linked to sequences from the end of the S component. The major defective genome species consisted of repeat units which were 4.9 x 10(6) in molecular weight and contained a specific deletion within the U(L) segment. These tsLB2 defective genomes were stable through more than 35 sequential virus passages. The ratios of defective virus genomes to helper virus genomes present in different passages fluctuated in synchrony with the capacity of the passages to interfere with standard virus replication. Cells infected with passages enriched for defective genomes overproduced the infected cell polypeptide number 8, which had previously been mapped within the U(L) sequences present in the tsLB2 defective genomes. In contrast, the synthesis of most other infected cell polypeptides was delayed and reduced. The abundant synthesis of infected cell polypeptide number 8 followed the beta regulatory pattern, as evident from kinetic studies and from experiments in which cycloheximide, canavanine, and phosphonoacetate were used. However, in contrast to many beta (early) and gamma (late) viral polypeptides, the synthesis of infected cell polypeptide number 8 was only minimally reduced when cells infected with serially passaged tsLB2 were incubated at 39 degrees C. The tsLB2 mutation had previously been mapped within the domains of the gene encoding infected cell polypeptide number 4, the function of which was shown to be required for beta and gamma viral gene expression. It is thus possible that the tsLB2 mutation affects the synthesis of only a subset of the beta and gamma viral polypeptides. An additional polypeptide, 74.5 x 10(3) in molecular weight, was abundantly produced in cells infected with a number of tsLB2 passages. This polypeptide was most likely expressed from truncated gene templates within the most abundant, deleted repeats of tsLB2 defective virus DNA.

Sequence analysis of cDNA's derived from the RNA of Sindbis virions and of defective interfering particles.

Sindbis virus generates defective interfering (DI) particles during serial high-multiplicity passage in cultured cells. These DI particles inhibit the replication of infectious virus and can be an important factor in the establishment and maintenance of persistent infection in BHK cells. In an effort to understand how these DI particles are generated and how they interfere with the replication of standard virus, we performed a partial sequence analysis of the RNA obtained from two independently isolated populations of DI particles and from two Sindbis virus variants and compared these with the RNA of the parental wild-type virus. The 3'-terminal regions of the RNAs were sequenced by the dideoxy chain terminating method. Internal regions of the RNA were examined by restriction endonuclease digestion of cDNA's made to the various RNAs and by direct chemical sequencing of 5' end-labeled restriction fragments from cDNA made to the DI RNAs. One of the variant viruses examined was originally derived from cells persistently infected with Sindbis virus for 16 months and is resistant to interference by the DI strains used. In the 3'-terminal region of the RNA from this variant, only two base changes were found; one of these occurs in the 20-nucleotide 3'-terminal sequence which is highly conserved among alphaviruses. The DI RNA sequences were found to have been produced not by a single deletional event, but by multiple deletion steps combined with sequence rearrangements; all sequences examined are derived from the plus strand of Sindbis virion RNA. Both DI RNAs had at least 50 nucleotides of wild-type sequence conserved at the 3' terminus; in addition, they both contained conserved and perhaps amplified sequences derived from the non-26S region of the genome which may be of importance in their replication and interference ability.

Generation of defective interfering particles of vesicular stomatitis virus in Aedes albopictus cells

The serial undiluted passage of clonally purified vesicular stomatitis virus (VSV) in Aedes albopictus cells resulted in a rapidly declining titer of infectious virus. VSV AP2 (a stock derived after two such passages) contained slowly sedimenting truncated (T) particles which were interfering and noninfectious. Whereas a VSV stock produced by three undiluted passages in BHK cells (VSV BP3) interfered with the replication of standard virus (VSV STD) equally well in A. albopictus and in BHK cells, VSV AP2 interfered efficiently only in the former. These patterns of interference were also observed when gradient purified T particles were tested. When VSV AP2 or VSV BP3 was passaged once in the heterologous cell type the pattern of interference was reversed showing that these defective interfering (DI) particles could be phenotypically modified in such a way as to influence their specificity of interference with VSV STD. When viral RNA synthesis was studied in cells coinfected with VSV STD and VSV AP2 or VSV BP3, the following observations were made: (1) interference was correlated with a decrease in the synthesis of 42 S VSV STD genome RNA; (2) although VSV AP2 DI particles interfered well in A. albopictus cells but only poorly in BHK cells the DI RNA genomes of VSV AP2 were replicated equally well in both cell types. (3) The major DI particles in VSV AP2 contained “snap-back” RNA composed of covalently linked sequences of positive and negative polarity. By testing the effect of RNase on these RNAs it was concluded that they contained from 90 to 95% selfcomplementary sequences. Snap-back RNA was not readily detectable in the DI particles of VSV BP3.

Alterations in virus protein synthesis and capsid production in infection with DI particles of herpesvirus.

High multiplicity, undiluted passage of equine herpesvirus type 1 (EHV-1) in L-M cells resulted in the rapid production of virus particles whose genome was genetically less complex, contained more reiterated DNA sequences and exhibited a greater buoyant density (rho = 1.724 g/ml) than the DNA (rho = 1.716 g/ml) of standard virus. These data and the finding that these particles inhibited the replication of standard virus in interference assays confirmed that these were defective interfering (DI) particles (Henry et al. 1979). Additional evidence for this has been obtained from the pattern of cyclic fluctuation in infectious virus titre through 17 serial passages as well as from the pronounced variation in the particle to plaque ratio for each passage. Total particle production was markedly reduced in cells infected with virus preparations containing DI particles and quantification of major cell-associated EHV-1 capsid species by electron microscopy and analysis in Renografin density gradients indicated that this reduction occurred at the level of capsid assembly. Although total capsid production was reduced in cells infected with DI particle preparations, the synthesis of I (immature) capsids increased relative to that of L (empty) capsids and these alterations in the assembly of capsid species could be related to changes in the synthesis of capsid proteins. In cells infected with EHV-1 preparations rich in DI particles, the synthesis of major capsid protein 150000 was greatly reduced, whereas core protein 46000, a major component of I capsids, was overproduced as compared to standard virus infection. Capsids produced in cells infected with virus preparations rich in DI particles were identical in polypeptide composition to those made in standard virus infection.

Generation of defective interfering particles in picornaviruses

We have extensively searched for defective interfering (DI) particles in several picornaviruses. Contrary to expectations, only the originally described poliovirus type I strain ( C. N. Cole, D. Smoller, E. Wimmer, and D. Baltimore, 1971, J. Virol. 7, 478–485) gave rise to DI separable in CsCl density gradients. However, by using high resolution gel analysis of glyoxal-denaturated RNAs, we detected small deletions (4–6%) in the virus particle genomes from long-term undiluted passage of mengovirus and two independent strains of poliovirus type I. Virus stocks enriched for these smaller RNAs were shown to interfere with the replication of standard virus. DI generation appears to be strain dependent since a third isolate of poliovirus type I does not give rise to detectable genome deletions even after 100 passages. Furthermore, a cloned stock of the Cole et al. strain regenerates a low density DI as observed originally. We also present evidence suggesting that host cell factors modulate both generation and interference level of a picornavirus DI (mengovirus). Vaccine reference strains of poliovirus did not contain detectable deleted genomes in contrast to a preliminary report from this laboratory. These studies have important implications for the possible role of DI in human or other picornavirus infections.

Role of virus variants and cells in maintenance of persistent infection by measles virus.

Hamster embryo fibroblasts persistently infected with a derivative of the Schwarz vaccine strain of measles virus spontaneously released virus particles with an average buoyant density considerably lower than that of the parental virus. The released virus contained all of the measles virus structural proteins and interfered with replication of standard virus. All of the virus structural proteins were associated with a membrane-free cytoplasmic extract from the persistently infected cells. Membrane-free cytoplasmic extracts prepared from Vero cells lytically infected with Schwarz strain measles contained little or no virus envelope structural protein. Maintenance of persistent infection may involve both the presence of virus variants and a defect in the ability of the infected cell to replicate the virus efficiently.

Generation of defective interfering particles of Semliki Forest virus in a clone of Aedes albopictus (mosquito) cells.

Serial undiluted passage of Semliki Forest virus in a clone of Aedes albopictus cells resulted in a marked decrease in infectious virus yields due to the generation and accumulation of defective interfering particles. Virus from the third passage had a high particle/infectivity ratio and interfered specifically with homologous but not heterologous standard virus replication. Two RNA species of molecular weights 0.78 X 10(6) and 0.61 X 10(6) were the major RNA components of purified passage 4 virus. These RNA species were also the predominant virus RNA species detected in cells infected with passage 3 virus. Synthesis of standard virus RNA and virus-specified protein was much reduced in passage 3 virus-infected cells. Interference with standard virus replication and the synthesis of large amounts of defective interfering RNA were also observed in chicken embryo cells infected with passage 3 virus from mosquito cells.

Homologous interference mediated by defective interfering influenza virus derived from a temperature-sensitive mutant of influenza virus.

A temperature-sensitive group II mutant of influenza virus, ts-52, with a presumed defect in viral RNA synthesis, readily produced von Magnus-type defective interfering virus (DI virus) when passed serially (four times) at high multiplicity in MDBK cells. The defective virus (ts-52 DI virus) had a high hemagglutinin and a low infectivity titer, and strongly interfered with the replication of standard infectious viruses (both ts-52 and wild-type ts+) in co-infected cells. Progeny virus particles produced by co-infection of DI virus and infectious virus were also defective and also had low infectivity, high hemagglutinating activity, and a strong interfering property. Infectious viruses ts+ and ts-52 were indistinguishable from ts-52 DI viruses by sucrose velocity or density gradient analysis. Additionally, these viruses all possessed similar morphology. However, when the RNA of DI viruses was analyzed by use of polyacrylamide gels containing 6 M urea, there was a reduction in the amount of large RNA species (V1 to V4), and a number of new smaller RNA species (D1 to D6) with molecular weights ranging from 2.9 X 10(5) to 1.05 X 10(5) appeared. Since these smaller RNA species (D1 to D6) were absent in some clones of infectious viruses, but were consistently associated with DI viruses and increased during undiluted passages and during co-infection of ts-52 with DI virus, they appeared to be a characteristic of DI viruses. Additionally, the UV target size of interfering activity and infectivity of DI virus indicated that interfering activity was 40 times more resistant to UV irradiation than was infectivity, further implicating small RNA molecules in interference. Our data suggest that the loss of infectivity observed among DI viruses may be due to nonspecific loss of a viral RNA segment(s), and the interfering property of DI viruses may be due to interfering RNA segments (DIRNA, D1 to D6). ts-52 DI virus interfered with the replication of standard virus (ts+) at both permissive (34 degrees C) and nonpermissive temperatures. The infectivity of the progeny virus was reduced to 0.2% for ts+ and 0.05% for ts-52 virus without a reduction in hemagglutinin titer. Interference was dependent on the concentration of DI virus. A particle ratio of 1 between DI virus (0.001 PFU/cell) and infectious virus (1.0 PFU/cell) produced a maximal amount of interference. Infectious virus yield was reduced 99.9% without any reduction of the yield of DI viruses Interference was also dependent on the time of addition of DI virus. Interference was most effective within the first 3 h of infection by infectious virus, indicating interference with an early function during viral replication.

Induction and biological properties of defective interfering particles of rabies virus.

A method for obtaining large quantities of defective interfering (DI) rabies virus particles that fulfill all the criteria delineated by Huang and Baltimore (1970) is described. The purified rabies DI virion was found to be much shorter (60 to 80 nm) than the complete virion (180 nm) and to have a viral genome of about half the size of normal rabies RNA but with all of the structural proteins of standard virions. Rabies DI virions were noninfectious for both cells in culture and for animals. As determined by in vitro and in vivo techniques, interference with the replication of standard virus was specific to rabies virus. The possible role of rabies DI virion in the pathogenicity of rabies virus infection and in the establishment of attenuated strains for use as live rabies vaccines is discussed.