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Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Correspondence
Nigel J. Dimmock
n.j.dimmock{at}warwick.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Huang & Baltimore, 1970, 1977; Holland, 1990) and, when there is a high ratio of DI : infectious virus, can totally abrogate detectable genome synthesis, protein synthesis and infectious virus production (e.g. Barrett et al., 1981; Barrett & Dimmock, 1985). In cell culture, there is a quantitative relationship between the amount of infectious virus and DI virus produced – initially, the infectious genome replicates both itself and the defective genome, but the small DI genome gives it a replicative advantage and eventually, when a high ratio of DI virus : infectious virus prevails, interference takes place. The resulting fall in helper virus leads to a loss of DI virus and reascendency of infectivity. This can lead to cycling, with peaks and troughs of infectious virus load (von Magnus, 1954; Roux et al., 1991).
In view of the success of DI viruses at interfering with infectious virus production in vitro, it is remarkable that there have been so few reports of DI viruses protecting adult animals from clinical disease in vivo (reviewed by Barrett & Dimmock, 1986). While negative reports are rarely published, it seems likely that this paucity of information means that the DI viruses tested failed to protect against disease in adult animal models. If the reasons for this failure could be established, a way forward might be found to establish a new generation of DI virus-based antivirals.
A notable exception with well-documented ability to protect adult animals is DI Influenza A virus. Influenza virus has a single-stranded RNA genome comprising eight separate segments (Lamb & Krug, 1996). Preparations of DI influenza virus usually contain several to many DI RNAs (see below). These are derived mainly from virion segments 1–3, but also from other segments (Jennings et al., 1983; Nayak et al., 1985, 1989; Noble & Dimmock, 1995; Duhaut & Dimmock, 1998). One DI virus preparation was found to contain more than 47 different DI RNA sequences (Duhaut & Dimmock, 1998). An RNA segment gives rise to several unique DI RNAs because of variation in the position of the internal breakpoints. DI influenza virus protects mice (reviewed by Dimmock, 1996) and ferrets (Mann et al., 2006) from Influenza A virus-induced respiratory disease. The mechanism by which this occurs is not clear, but requires the integrity of the DI RNA, as protection is abrogated by UV irradiation or 
-propiolactone (Dimmock et al., 1986; Noble & Dimmock, 1994) and may involve the ability of DI RNAs to compete for a limited supply of infectious virus-synthesized trans-acting factors, such as components of the RNA replication machinery, that a DI virus possessing defective RNAs 1, 2 or 3 would not be able to synthesize. Almost certainly, DI virus readjusts the balance between the infection and the host-defence system, so that the animal is able to recover from infection with a lower severity of disease or, better still, no disease symptoms at all. However, clinical benefit is seen only when DI influenza virus is administered before, or at the same time as, infection; DI virus has little therapeutic activity (Dimmock et al., 1986; Noble & Dimmock, 1994; L. McLain and N. J. Dimmock, unpublished data).
Influenza A viruses have a common replication mechanism, as shown by their ability to interact genetically and form viable recombinants (Webster et al., 1992); thus, a DI genome should be replicated by all influenza A strains, regardless of subtype, and inhibit their replication. This is potentially important, as it means that DI virus could act as a generic antiviral for Influenza A virus. However, it is reported here that a DI influenza A virus preparation that protected mice from lethal infection with the A/PR8 (H1N1) and A/WSN (H1N1) strains of influenza virus had no activity against clinical disease caused by an H2N2 virus. It transpired that this failure did not reside in any intrinsic inability of infectious virus to replicate the DI virus, or of DI genomes to interfere with infectious virus replication. Rather, the key finding was that the H2N2 virus needed a 300-fold higher mouse infectious dose to cause clinical disease than A/PR8 and that this high infectious virus : DI virus ratio prevented the interfering activity of DI virus from being expressed. This finding underlines the importance of the infectious dose rather than the disease-causing dose of virus and may help in establishing systems to study the in vivo antiviral activity of DI viruses other than influenza virus.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Infectivity titrations.
Cell culture, embryonated chicken egg and mouse infectivity titres were determined for each virus. Virus was plaque-assayed in MDCK cells under agar by standard procedures and plaques were counted after 4 days incubation. Eggs were inoculated with limit-diluted virus and incubated for 3 days. Virus-positive eggs were identified by haemagglutination by allantoic fluids. Mice were inoculated as described above with limit-diluted virus. Three days later, mice were killed and ground lungs from individual mice were inoculated into eggs. The presence of virus was determined by haemagglutination with chicken red blood cells. Egg and mouse infectivity titres were calculated according to Spearman–Kärber (Kärber, 1968).
DI virus.
The DI virus used in this study was originally produced from A/equine/Newmarket/7339/79 (A/EQV, H3N8) by three high-multiplicity, overnight passages of infectious virus in embryonated chicken eggs [approx. 102 haemagglutination units (HAU) per egg]. DI virus was purified by differential centrifugation, standardized by haemagglutination titration and stored in liquid nitrogen. Such preparations contain several different DI RNAs from different genomic segments and protect mice from lethal infection with various subtypes of Influenza A virus (Noble & Dimmock, 1994, 1995; Duhaut & Dimmock, 1998; Noble et al., 2004). However, sequencing studies showed that the population of DI RNAs in egg-grown virus differed markedly from that found in the lungs of mice infected with that DI virus (Duhaut & Dimmock, 1998), suggesting that not all DI RNAs replicated well in the mouse. As it seemed logical that only DI RNAs that were replicated in the mouse were likely to be antiviral, a mouse-adapted A/EQV DI virus was made by passaging DI and helper A/EQV three times in mice via undiluted lung homogenates. Eggs were then inoculated with the final lung homogenate to amplify these DI RNAs. Whilst this was successful, there was still a problem with poor A/EQV growth so, to improve yields, third mouse-passage A/EQV DI RNAs were rescued by using a high-growing A/PR8 strain, also in mice. Before inoculation, the EQV DI virus preparation was rendered non-infectious by irradiation with a critical dose (40 s) of UV light. The UV inactivates infectivity that would confuse the infectious dose given to the mice. DI RNA and activity are not affected significantly, as it has a smaller UV target size (approx. 400 nt) than that of the infectious genome (13 600 nt). The lamp was calibrated by inactivating A/PR8 infectivity. Lung homogenate was then passaged in eggs three times at high m.o.i. The resulting passage 3 virus was confirmed as a reassortant by the presence of A/PR8, but not A/EQV haemagglutinin (HA), and the presence of A/EQV DI RNAs as determined by RT-PCR. This was designated mouse-passaged DI A/EQV(PR8) virus. This DI virus strongly protected mice from a lethal dose of A/PR8 or A/WSN influenza virus (see Results), whereas DI A/PR8 produced by passaging A/PR8 in parallel was only weakly protective (data not shown). Finally, DI virus was purified by differential centrifugation and pelleting through 10 % sucrose and was resuspended at 105 HAU ml–1 in PBS containing 0·1 % (w/v) BSA.
Animal inoculation.
C3H/He-mg (H-2k) mice were inoculated intranasally as described previously (Noble & Dimmock, 1994; Noble et al., 2004), with interfering but non-infectious DI virus, which was produced by UV irradiation for 40 s as described above. To control for any immune system-stimulating or receptor-blocking effects, other mice were inoculated in parallel with a ‘dead’ non-interfering preparation produced by prolonged UV irradiation (8 min). This inactivates both infectivity and interfering activity, but does not affect haemagglutination or neuraminidase activities. Specifically, mice (4-week-old; 16–20 g) were lightly anaesthetized with ether and a 20 µl inoculum was divided between the two nares. Inocula comprised active, non-infectious DI virus containing a defined dose of infectious influenza virus, UV-killed DI virus, active DI virus alone, or diluent. Infectious viruses were titrated in mice to determine a dose for each that caused comparable respiratory disease. Morbidity was assessed according to loss of weight and by previously described clinical criteria (Noble & Dimmock, 1994). Clinical criteria were scored quantitatively as follows: 1 point for each healthy mouse; 2 points for each mouse showing signs of malaise, including slight piloerection, slightly changed gait and increased ambulation; 3 points for each mouse showing signs of strong piloerection, constricted abdomen, changed gait, periods of inactivity, increased breathing rate and sometime râles; 4 points for each mouse with enhanced characteristics of the previous group, but showing little activity and becoming moribund (such mice were killed when it was clear that they would not survive); and 5 points for a dead mouse. To achieve parity, the total clinical score was divided by the number of mice in the experimental group. All viruses caused similar clinical disease, including lung consolidation. Experiments followed the guidelines of the UK Co-ordinating Committee for Cancer Research.
RT-PCR.
RNA was extracted from the lungs of one mouse by grinding with sterile sand in 4 ml TRIzol reagent (Invitrogen) and dissolved in 100 µl water. Aliquots of 5 µg RNA were reverse-transcribed in 20 µl reactions for 1 h at 42 °C, using a generic type A influenza RNA 1-specific primer (RNA1F, 5'-AGCGAAAGCAGGTCAAATATA-3'), complementary to the 3' terminus of the viral RNA (vRNA). RNA 1 encodes the PB2 protein component of the viral replicase. Aliquots (1·5 µl) of the reverse transcription reaction were then amplified by PCR using Taq DNA polymerase (MBI Fermentas) and generic primers specific for RNA 1 of Influenza A virus, RNA1F and RNA1R (5'-AGTAGAAACAAGGTCGTTTTTA-3', complementary to the 3' terminus of the cRNA), or primers specific for RNA 1 of A/EQV virus EQV1F (5'-CAAATATATTCAATATGGAG-3', complementary to nt 14–33 of vRNA) and EQV1R (5'-GGTCGTTTTTAAACAATTCA-3', complementary to nt 12–31 of the cRNA). EQV1F and EQV1R have mismatches compared with the equivalent sequences in A/PR8 and A/Jap.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The dose of A/PR8 was kept at 10 LD50. The progress of infection was monitored by daily weighing and clinical assessment (Fig. 1). Data show that mice infected with 10 LD50 A/PR8 were completely protected by DI virus, whereas those given UV-inactivated DI virus were all dead by day 10 (Fig. 1a–c). The same scenario was seen in mice infected with 10 LD50 WSN (data not shown). However, Fig. 1(d–f) shows that, despite the lower dose of A/Jap and increased application of DI virus, only marginal protection was observed: a slight sparing effect in the clinical scores of infected mice given DI treatment (Fig. 1f) and a reduction in the mortality between the infected groups treated with DI virus (2/11 or 18 %) or with inactivated DI virus (5/11 or 45 %) (Fig. 1e). The difference in weight loss between the infected groups treated with DI virus or with inactivated DI virus did not differ statistically (Fig. 1d). These data were highly reproducible and similar results were obtained with other preparations of DI virus. The lack of protection against A/Jap was unexpected, as influenza A viruses have a common replication mechanism (Webster et al., 1992) and should thus replicate all influenza A DI genomes and be inhibited by them. Possible explanations for the low inhibition of A/Jap by DI virus are that (a) A/Jap does not replicate the applied DI RNAs in mouse lung, (b) A/Jap replicates DI virus in the lung, but is insensitive to DI virus-mediated interference or (c) infection of mice by A/Jap requires a larger dose of infectious virus than infection with the other influenza viruses and thus interference by DI virus is ineffective.
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, lane 4). Further, reaction of the same RNA with A/EQV RNA-1-specific primers strongly amplified a smaller band of 340 bp, suggesting that the major 370 nt DI RNA band identified in lane 4 is derived specifically from A/EQV and not from A/PR8 or A/Jap (Fig. 2, lane 5). Thus, the replication systems of A/Jap and the DI virus are compatible and failure to replicate DI genomes is not the reason for the lack of protection from A/Jap-mediated disease. Finally, although only the presence of defective RNAs derived from RNA 1 is shown, it is likely that DI RNAs originating from other virion RNAs were also present (Duhaut & Dimmock, 1998).
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shows that A/Jap and A/PR8 had similar egg infectivity titres. The HAU reflects the total number of physical virus particles present and allows the calculation of specific infectivity (egg infectivity : HAU ratio). The ratios for A/Jap and A/PR8 were also very similar (Table 1). Infectivity was also determined by end-point titration in mice and, by this measure, A/Jap had fourfold-lower specific mouse infectivity than A/PR8 (Table 1). However, A/PR8 had a higher specific lethality than A/Jap. With this information, the level of infectivity required by A/Jap and A/PR8 to cause disease in mice was calculated and it was found that a lethal dose of A/Jap contained 2000-fold more egg infectious units and 300-fold more mouse infectious units than A/PR8 (Table 2). This suggests that DI virus did not protect mice against A/Jap because there is an unfavourably high infectious virus : DI virus ratio. The fact that a 10-fold dilution of DI virus abrogates protection against PR8-induced disease (Table 4) supports the notion of the importance of the DI virus : infectious virus ratio (Table 4).
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) or A/WSN (data not shown). As the dose of A/Jap was too low to cause disease, lung virus infectivity was the read-out. Lung samples were taken at 24 h post-infection when A/Jap infectivity first reached a plateau (data not shown). Table 3 shows that A/Jap lung infectivity was reduced by 100-fold in mice given active DI virus compared with those given UV-inactivated control DI virus. These data indicate that DI virus can interfere with multiplication of A/Jap in mice, provided that there is an appropriately high ratio of DI virus : infectious virus, a putative critical ratio.
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). WSN is thus the most virulent of the viruses used in this study. This situation allowed us to test the prediction that one-tenth of the DI virus dose used above should protect mice against the low amount of infectious A/WSN present in 10 LD50. This indeed proved to be the case and Table 4 shows that 20 % of mice given A/WSN plus active DI virus developed clinical disease, compared with 100 % of mice given A/WSN and inactivated DI virus. However, as predicted from the critical-dose hypothesis, the same low dose of DI virus did not protect mice against the 33-fold-higher infectious dose of A/PR8 that is required to cause disease (Table 4). Nonetheless, as expected, the low DI virus dose interfered significantly with the multiplication in the lung of a 100-fold-lower dose of A/PR8 (data not shown). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The rather surprising conclusion is that DI virus is most effective against virulent virus, i.e. virus that infects with the least number of virus particles.
Thus far, we have been unable to determine the protective ratio of defective genomes : infectious genomes, as spontaneously occurring preparations of DI Influenza A virus contain a large and varied population of defective RNA sequences with different deletion break points, which can originate from all eight segments of RNA (Noble & Dimmock, 1995; Duhaut & Dimmock, 1998). A further complication is that different DI RNA sequences may not interfere with the same efficiency (Thomson et al., 1998). What is needed for quantification is a cloned DI/infectious influenza virus preparation that contains a single, identifiable DI RNA. A prototype system has been described, but needs further development to make it a practical proposition (Duhaut & Dimmock, 2003). The varied nature of DI genome sequences in uncloned preparations also makes it difficult to determine how DI virus exerts protection. A recent review of the mechanism of action of DI RNAs points out that not all DI RNAs are protective and that protecting DI RNAs may act by competing for transactivating factors, sequestering a limited amount of virus product such as polymerase, prevention of protein–protein interactions or production of small interfering RNAs (Simon et al., 2004).
This study underlines the fundamental point that different virus strains have a unique infectious dose for each in vivo or in vitro assay system used and thus the infectious dose : disease-causing dose ratio in vivo is likely to be strain-unique. A priori, evolution probably disfavours any mutant that requires a large infectious dose, as infection is unlikely to produce a sufficiently high virus titre in body fluids and/or to transmit a large amount of virus to a new host. Thus, viruses would be expected to evolve a small infectious dose. This would be good news for any proposed DI-mediated prophylaxis, as only a minimal dose of DI virus would be required to abort clinical disease successfully.
Our manipulation of the DI virus used in this study is of some interest. This is the first time that a DI virus has been produced that is enriched for DI RNAs that are preferentially replicated by an animal and the first time that defective RNAs have been incorporated into a new helper virus. No selection pressure was available, but none was needed. Additionally, there seemed to be no problem in transferring defective RNA from a parent virus of one subtype (H3N8) to that of a virus of a different subtype (H1N1). This was all achieved by using simple, non-recombinant DNA technologies.
This study supports the hypothesis that DI Influenza A virus has antiviral activity against all influenza A viruses and further defines this relationship. On this basis, it is suggested that this generic activity should be considered as an additional measure to counter newly emerging and potentially catastrophic pandemic influenza A virus strains (Webster, 1997). Finally, our conclusion concerning the critical ratio of DI virus : infectious virus required for protection of animals may explain why so few in vivo models for the antiviral activity of DI viruses have been described and may help in devising new model systems for studying the prevention of virus diseases.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Duhaut, S. D. & Dimmock, N. J. (1998). Heterologous protection of mice from a lethal human H1N1 influenza A virus infection by H3N8 equine defective interfering virus: comparison of defective RNA sequences isolated from the DI inoculum and mouse lung. Virology 248, 241–253.[CrossRef][Medline]
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Mann, A., Marriott, A. C., Balasingam, S., Lambkin, R., Oxford, J. S. & Dimmock, N. J. (2006). Interfering vaccine (defective interfering influenza A virus) protects ferrets from influenza, and allows them to develop solid immunity to reinfection. Vaccine (in press).
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Noble, S. & Dimmock, N. J. (1994). Defective interfering type A equine influenza virus (H3N8) protects mice from morbidity and mortality caused by homologous and heterologous subtypes of influenza A virus. J Gen Virol 75, 3485–3491.
Noble, S. & Dimmock, N. J. (1995). Characterization of putative defective interfering (DI) A/WSN RNAs isolated from the lungs of mice protected from an otherwise lethal respiratory infection with influenza virus A/WSN (H1N1): a subset of the inoculum DI RNAs. Virology 210, 9–19.[CrossRef][Medline]
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Received 10 November 2005;
accepted 8 January 2006.
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, DI virus+infectious virus; 
, UV-inactivated DI virus+infectious virus; 
, DI virus alone. Results are representative of twosuch experiments.