Impact of defective interfering particles on virus replication and antiviral host response in cell culture-based influenza vaccine production

During the replication of influenza viruses, defective interfering particles (DIPs) can be generated. These are noninfectious deletion mutants that require coinfection with a wild-type virus but interfere with its helper virus replication. Consequently, coinfected cells mainly produce DIPs. Little is known about how such noninfectious virus particles affect the virus yield of cell culture-based influenza vaccine production. We compared infections of Madin-Darby canine kidney cells with two seed virus preparations of the influenza virus strain A/Puerto Rico/8/34 that contain different amounts of DIPs. A combination of conventional RT-PCR, RT-qPCR, and flow cytometry revealed that DI genomes indeed strongly accumulate in coinfected cells and impede the viral RNA synthesis. Additionally, cells infected at the higher DIP concentration showed a stronger antiviral response characterized by increased interferon-β expression and apoptosis induction. Furthermore, in the presence of DIPs, a significant fraction of cells did not show any productive accumulation of viral proteins at all. Together, these effects of DIPs significantly reduce the virus yield. Therefore, the accumulation of DIPs should be avoided during influenza vaccine production which can be achieved by quality controls of working seed viruses based on conventional RT-PCR. The strategy for the depletion of DIPs presented here can help to make cell culture-based vaccine production more reliable and robust.     

Cloned Defective Interfering Influenza Virus Protects Ferrets from Pandemic 2009 Influenza A Virus and Allows Protective Immunity to Be Established

Influenza A viruses are a major cause of morbidity and mortality in the human population, causing epidemics in the winter, and occasional worldwide pandemics. In addition there are periodic outbreaks in domestic poultry, horses, pigs, dogs, and cats. Infections of domestic birds can be fatal for the birds and their human contacts. Control in man operates through vaccines and antivirals, but both have their limitations. In the search for an alternative treatment we have focussed on defective interfering (DI) influenza A virus. Such a DI virus is superficially indistinguishable from a normal virus but has a large deletion in one of the eight RNAs that make up the viral genome. Antiviral activity resides in the deleted RNA. We have cloned one such highly active DI RNA derived from segment 1 (244 DI virus) and shown earlier that intranasal administration protects mice from lethal disease caused by a number of different influenza A viruses. A more cogent model of human influenza is the ferret. Here we found that intranasal treatment with a single dose of 2 or 0.2 µg 244 RNA delivered as A/PR/8/34 virus particles protected ferrets from disease caused by pandemic virus A/California/04/09 (A/Cal; H1N1). Specifically, 244 DI virus significantly reduced fever, weight loss, respiratory symptoms, and infectious load. 244 DI RNA, the active principle, was amplified in nasal washes following infection with A/Cal, consistent with its amelioration of clinical disease. Animals that were treated with 244 DI RNA cleared infectious and DI viruses without delay. Despite the attenuation of infection and disease by DI virus, ferrets formed high levels of A/Cal-specific serum haemagglutination-inhibiting antibodies and were solidly immune to rechallenge with A/Cal. Together with earlier data from mouse studies, we conclude that 244 DI virus is a highly effective antiviral with activity potentially against all influenza A subtypes.     

Defective Interfering Influenza Virus RNAs: Time To Reevaluate Their Clinical Potential as Broad-Spectrum Antivirals?

Defective interfering (DI) RNAs are highly deleted forms of the infectious genome that are made by most families of RNA viruses. DI RNAs retain replication and packaging signals, are synthesized preferentially over infectious genomes, and are packaged as DI virus particles which can be transmitted to susceptible cells. Their ability to interfere with the replication of infectious virus in cell culture and their potential as antivirals in the clinic have long been known. However, until now, no realistic formulation has been described. In this review, we consider the early evidence of antiviral activity by DI viruses and, using the example of DI influenza A virus, outline developments that have led to the production of a cloned DI RNA that is highly active in preclinical studies not only against different subtypes of influenza A virus but also against heterologous respiratory viruses. These data suggest the timeliness of reassessing the potential of DI viruses as a novel class of antivirals that may have general applicability.     

Influenza virus protecting RNA: an effective prophylactic and therapeutic antiviral

Another influenza pandemic is inevitable, and new measures to combat this and seasonal influenza are urgently needed. Here we describe a new concept in antivirals based on a defined, naturally occurring defective influenza virus RNA that has the potential to protect against any influenza A virus in any animal host. This “protecting RNA” (244 RNA) is incorporated into virions which, although noninfectious, deliver the RNA to those cells of the respiratory tract that are naturally targeted by infectious influenza virus. A 120-ng intranasal dose of this 244 protecting virus completely protected mice against a simultaneous challenge of 10 50% lethal doses with influenza A/WSN (H1N1) virus. The 244 virus also protected mice against strong challenge doses of all other subtypes tested (i.e., H2N2, H3N2, and H3N8). This prophylactic activity was maintained in the animal for at least 1 week prior to challenge. The 244 virus was 10- to 100-fold more active than previously characterized defective influenza A viruses, and the protecting activity was confirmed to reside in the 244 RNA molecule by recovering a protecting virus entirely from cloned cDNA. There was a clear therapeutic benefit when the 244 virus was administered 24 to 48 h after a lethal challenge, an effect which has not been previously observed with any defective virus. Protecting virus reduced, but did not abolish, replication of challenge virus in mouse lungs during both prophylactic and therapeutic treatments. Protecting virus is a novel antiviral, having the potential to combat human influenza virus infections, particularly when the infecting strain is not known or is resistant to antiviral drugs.     

Generation of a purely clonal defective interfering influenza virus

Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals.     

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.     

Sequence Analysis of In Vivo Defective Interfering-Like RNA of Influenza A H1N1 Pandemic Virus

Influenza virus defective interfering (DI) particles are naturally occurring noninfectious virions typically generated during in vitro serial passages in cell culture of the virus at a high multiplicity of infection. DI particles are recognized for the role they play in inhibiting viral replication and for the impact they have on the production of infectious virions. To date, influenza virus DI particles have been reported primarily as a phenomenon of cell culture and in experimentally infected embryonated chicken eggs. They have also been isolated from a respiratory infection of chickens. Using a sequencing approach, we characterize several subgenomic viral RNAs from human nasopharyngeal specimens infected with the influenza A(H1N1)pdm09 virus. The distribution of these in vivo-derived DI-like RNAs was similar to that of in vitro DIs, with the majority of the defective RNAs generated from the PB2 (segment 1) of the polymerase complex, followed by PB1 and PA. The lengths of the in vivo-derived DI-like segments also are similar to those of known in vitro DIs, and the in vivo-derived DI-like segments share internal deletions of the same segments. The presence of identical DI-like RNAs in patients linked by direct contact is compatible with transmission between them. The functional role of DI-like RNAs in natural infections remains to be established.     

Mutational analysis of cis-acting RNA signals in segment 7 of influenza A virus

The genomic viral RNA (vRNA) segments of influenza A virus contain specific packaging signals at their termini that overlap the coding regions. To further characterize cis-acting signals in segment 7, we introduced synonymous mutations into the terminal coding regions. Mutation of codons that are normally highly conserved reduced virus growth in embryonated eggs and MDCK cells between 10- and 1,000-fold compared to that of the wild-type virus, whereas similar alterations to nonconserved codons had little effect. In all cases, the growth-impaired viruses showed defects in virion assembly and genome packaging. In eggs, nearly normal numbers of virus particles that in aggregate contained apparently equimolar quantities of the eight segments were formed, but with about fourfold less overall vRNA content than wild-type virions, suggesting that, on average, fewer than eight segments per particle were packaged. Concomitantly, the particle/PFU and segment/PFU ratios of the mutant viruses showed relative increases of up to 300-fold, with the behavior of the most defective viruses approaching that predicted for random segment packaging. Fluorescent staining of infected cells for the nucleoprotein and specific vRNAs confirmed that most mutant virus particles did not contain a full genome complement. The specific infectivity of the mutant viruses produced by MDCK cells was also reduced, but in this system, the mutations also dramatically reduced virion production. Overall, we conclude that segment 7 plays a key role in the influenza A virus genome packaging process, since mutation of as few as 4 nucleotides can dramatically inhibit infectious virus production through disruption of vRNA packaging.     

Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production

Background  

In cell culture-based influenza vaccine production the monitoring of virus titres and cell physiology during infection is of great importance for process characterisation and optimisation. While conventional virus quantification methods give only virus titres in the culture broth, data obtained by fluorescence labelling of intracellular virus proteins provide additional information on infection dynamics. Flow cytometry represents a valuable tool to investigate the influences of cultivation conditions and process variations on virus replication and virus yields.     

Airborne Influenza A Is Detected in the Personal Breathing Zone of Swine Veterinarians

The 2009 H1N1 pandemic emphasized a need to evaluate zoonotic transmission of influenza A in swine production. Airborne influenza A virus has been detected in swine facilities during an outbreak. However, the personal exposure of veterinarians treating infected swine has not been characterized. Two personal bioaerosol samplers, the NIOSH bioaerosol sampler and the personal high-flow inhalable sampler head (PHISH), were placed in the breathing zone of veterinarians treating swine infected with either H1N1 or H3N2 influenza A. A greater number of viral particles were recovered from the NIOSH bioaerosol sampler (2094 RNA copies/m³) compared to the PHISH sampler (545 RNA copies/m³). In addition, the majority of viral particles were detected by the NIOSH bioaerosol sampler in the >4 μm size fraction. These results suggest that airborne influenza A virus is present in the breathing zone of veterinarians treating swine, and the aerosol route of zoonotic transmission of influenza virus should be further evaluated among agricultural workers.     

Interference among defective interfering particles of vesicular stomatitis virus

Three defective interfering (DI) particles of vesicular stomatitis virus (VSV), all derived from the same parental standard San Juan strain (Indiana serotype), were used in various combinations to infect cells together with the parental virus. The replication of their RNA genomes in the presence of other competing genomes was described by the hierarchical sequence: DI 0.52 particles greater than DI 0.45 particles less than or equal to DI-T particles greater than standard VSV. The advantage of one DI particle over another was not due simply to multiplicity effects nor to the irreversible occupation of limited cellular sites. Interference, however, did correlate with a change in the ratio of plus and minus RNA templates that accumulated intracellularly and with the presence of new sequences at the 3' end of the DI genomes. DI 0.52 particles contained significantly more nucleotides at the 3' end that were complementary to those at the 5' end of its RNA than did DI-T or DI 0.45 particles. The first 45 nucleotides at the 3' ends of all of the DI RNAs were identical. VSV and its DI particles can be separated into three classes, depending on their terminal RNA sequences. These sequences suggest two mechanisms, one based on the affinity of polymerase binding and the other on the affinity of N-protein binding, that may account for interference by DI particles against standard VSV and among DI particles themselves.     

A Naturally Occurring Variant of Porcine Mx1 Associated with Increased Susceptibility to Influenza Virus In Vitro

Mx1 has been implicated in resistance to the influenza virus. We have now identified four alleles of the Mxl gene in domesticated breeds of pigs. Two of the alleles encode deletion variants (a 3-bp deletion in exon 13 and an 11-bp deletion in exon 14), which might be expected to interfere with Mx activity. The porcine Mxl genes corresponding to wild type, the 3-bp deletion mutant, and the 11-bp deletion mutant were cloned and expressed in NIH3T3 cells, and the antiviral activity for influenza virus was assayed. Virus yield was observed to be 10–100-fold greater with the 11-bp deletion allele than that for wild type and the 3-bp deletion alleles. The results suggest that the 11-bp deletion type is lacking antiviral activity able to contribute to the interference of influenza virus replication.     

Baculovirus Mediated Production of Infectious Hepatitis C Virus in Human Hepatoma Cells Stably Expressing T7 RNA Polymerase

Although much has been learned about Hepatitis C virus (HCV), research progress has been hindered by the lack of a suitable cell culture system supporting its replication. Recently, a unique HCV strain JFH1 has been found to replicate efficiently in cell culture with production of infectious HCV (HCVcc). Baculovirus vectors were found to be efficient delivery vehicles and a HBV recombinant baculovirus/HepG2 system efficiently delivered the HBV genome into HepG2 resulting in HBV replication. In this study, we developed a recombinant baculovirus expression system to generate infectious HCV particles in hepatoma cell line Huh7-lunetT7 by using cDNA from the HCV JFH1 genotype. Results show that HCV positive, negative RNA strands and proteins were produced in this system. Furthermore, HCV particles were produced and secreted into the culture medium. Sucrose density gradient centrifugation of the culture medium revealed co-localization of HCV RNA and structural proteins in the fraction with a density of 1.08–1.13 g/ml. Electron microscopy (EM) showed viral particles approximately 55 nm in diameter, which could be recognized by anti-HCV E2 antibodies. Real-time RT-PCR detected that the level of HCV vRNA in the supernatant was 10⁷ copies/ml at 72 h post-transduction (hpt). In addition, the JFH1 virus produced by the recombinant baculovirus was confirmed to be infectious in vitro. In summary, this system provides a novel tool not only for the analysis of the replication and pathogenesis of HCV but also to screen for potential therapeutic targets.     

Defective interfering viral particles in acute dengue infections

While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles have referred to studies in vitro, there is some evidence for the presence of DI particles in chronic viral infections in vivo. In this study, short fragments of dengue virus (DENV) RNA containing only key regulatory elements at the 3' and 5' ends of the genome were recovered from the sera of patients infected with any of the four DENV serotypes. Identical RNA fragments were detected in the supernatant from cultures of Aedes mosquito cells that were infected by the addition of sera from dengue patients, suggesting that the sub-genomic RNA might be transmitted between human and mosquito hosts in defective interfering (DI) viral particles. In vitro transcribed sub-genomic RNA corresponding to that detected in vivo could be packaged in virus like particles in the presence of wild type virus and transmitted for at least three passages in cell culture. DENV preparations enriched for these putative DI particles reduced the yield of wild type dengue virus following co-infections of C6-36 cells. This is the first report of DI particles in an acute arboviral infection in nature. The internal genomic deletions described here are the most extensive defects observed in DENV and may be part of a much broader disease attenuating process that is mediated by defective viruses.     

Primary structure of poliovirus defective-interfering particle genomes and possible generation mechanisms of the particles

The genomes of defective-interfering (DI) particles derived from the Sabin strain of type 1 poliovirus (PV1(Sab] were characterized by nuclease S1 mapping using complementary DNA (cDNA) copies of PV1(Sab) genome as probes. The results demonstrated variety in the size and location of the deletions, which were compatible with our previous prediction. The results further indicated that the locations of the deletions were limited within the internal genome region encoding viral capsid proteins and that the deletion sites were clustered in certain areas on the genome. Sequence analysis of a number of cloned cDNAs to the DI genomes revealed that every DI genome retained the correct reading frame for viral protein synthesis. These results strongly suggested that one or all of the viral non-structural proteins might be cis-acting at least at a certain stage in viral replication. A computer search for secondary structures with regard to the deletion sites provided a possible common structure from which, supported by sequences existing on the plus or minus RNA strand of PV1(Sab), deletion regions looped out from the remaining sequences. Replicase might, therefore, skip these transiently formed loop structures with certain frequencies, resulting in the generation of DI genomes. This model could also be considered as a model for genetic recombination in these RNA genomes. Possible "supporting sequences" were also found for every rearranged site on the RNAs of influenza virus and sindbis virus. Thus, we propose a new copy-choice model, designated the "supporting sequence-loop model", for the generation of rearrangements occurring on single-stranded RNA genomes.     

Defective Viral Genomes: Critical Danger Signals of Viral Infections

Viruses efficiently block the host antiviral response in order to replicate and spread before host intervention. The mechanism initiating antiviral immunity during stealth viral replication is unknown, but recent data demonstrate that defective viral genomes generated at peak virus replication are critical for this process in vivo. This article summarizes the supporting evidence and highlights gaps in our understanding of the mechanisms and impact of immunostimulatory defective viral genomes generated during natural infections.