This discussion attempts to present a simple explanation of the current theory of error catastrophe and why it does not herald a paradigm shift in antiviral strategy. In the main text of the paper, the workings of a simple model of error catastrophe are examined to demonstrate what actually causes error catastrophe. The Appendix contains a more detailed discussion of the original error threshold model of Eigen and Schuster and how it applies to a viral quasispecies. RNA viruses are said to replicate at the edge of “error catastrophe” (18). Error catastrophe is a term coined to describe the supposed inability of a genetic element to be maintained in a population as the fidelity of its replication machinery decreases beyond a certain threshold value. Error catastrophe has been invoked as a theoretical basis for treatment of viral infection with drugs that would push the error rate for copying of the viral genome beyond this threshold (1, 4, 5, 6, 7, 9, 10, 14, 15, 16, 17, 19, 22, 25, 26, 28, 29, 40). Numerous publications aimed at the detection of virus extinction by error catastrophe induced by viral mutagens have appeared in recent years (8, 11, 12, 13, 24, 27, 33, 34, 35, 36, 38, 39, 46, 47). The catastrophic effect of high error rates was originally predicted in a mathematical model by Eigen and Schuster (20), in which a master genetic sequence replicated in competition with a collection of variants generated by errors in replication of the master sequence. In the simplest versions of the model (41), the variants all typically have a lower replication rate than the master sequence, and the effect of their replication errors is to convert one variant into another. When the distribution of genomes in such a replicating system was calculated to steady state, it was found that beyond a threshold error rate the master sequence effectively disappeared, becoming no more frequent than any single variant sequence. Eigen and Schuster referred to this hypothetical redistribution of the genetic information of the system as an error catastrophe (not to be confused with the theory of ageing that is also called error catastrophe [30, 31, 32]). Various treatments of the basic model have appeared in the literature since publication of Eigen and Schuster's original paper (2, 3, 21, 42, 42, 44, 45). We present here an examination of the theoretical basis for error catastrophe as predicted by the accepted mathematical simulations. For this purpose, we have constructed our own relatively simple model, based on ordinary differential equations, that reproduces error catastrophe. Using this model, we show that an error threshold is predicted to occur solely because of the implausible proposition that all progeny genomes that are not the master sequence continue to replicate at a finite rate no matter how many replication errors they contain, whereas replication of the master sequence is disqualified by a single error in the progeny genome. The disappearance of the master sequence at the error threshold is predicated on competition between the progeny and the master sequence to infinite time. We will show that, without the assumption that all mutants, no matter what their sequences, continue to replicate, mathematical models do not predict error catastrophe.

Chronic hepatitis B virus (HBV) infection is a major cause of morbidity and mortality worldwide, yet currently available therapies fail to provide long-term control of viral replication in most patients. Strategies to boost the weak virus-specific T-cell response typically found in patients with chronic hepatitis B have been proposed as a means of terminating persistent HBV infection. The potential problems arising from the stimulation of virus-specific immunity in a disease caused by a non-cytopathic virus, where viral control and liver injury are mediated by the immune system, are discussed. Furthermore, the concept of augmenting the HBV-specific T-cell response, which has previously been focused solely on quantitative issues, is expanded in the light of new findings of qualitative differences in the HBV-specific CD8 cell response.

Chronic hepadnavirus infections cause liver damage with ongoing death and regeneration of hepatocytes. In the present study we set out to quantify the extent of liver turnover by measuring the clonal proliferation of hepatocytes by using integrated viral DNA as a genetic marker for individual hepatocyte lineages. Liver tissue from woodchucks with chronic woodchuck hepatitis virus (WHV) infection was assayed for randomly integrated viral DNA by using inverse PCR. Serial endpoint dilution of viral-cell junction fragments into 96-well plates, followed by nested PCR and DNA sequencing, was used to determine the copy number of specific viral cell junctions as a measure of the clonal distribution of infected cell subpopulations. The results indicated that the livers contained a minimum of 100,000 clones of >1,000 cells containing integrated DNA, representing at least 0.2% of the hepatocyte population of the liver. Because cells with integrated WHV DNA comprised only 1-2% of total liver cells, it is likely that the total number of clones far exceeds this estimate, with as much as one-half of the liver derived from high copy clones of >1,000 cells. It may be inferred that these clones have a strong selective growth or survival advantage. The results provide evidence for a large amount of hepatocyte proliferation and selection having occurred during the period of chronic WHV infection ( approximately 1.5 years) in these animals.

ABSTRACT Inoculation of 3-day-old (3D) or 3-week-old (3W) ducklings with duck hepatitis B virus results in chronic or transient infection, respectively. We previously showed that rapid production of neutralizing antibody following inoculation of 3W ducklings prevents virus from spreading in the liver and leads to a transient infection (Y.-Y. Zhang and J. Summers, J. Virol. 78:1195-1201, 2004). In this study we further investigated early events of viral infection in both 3D and 3W ducks. We present evidence that a lower level of virus replication in the hepatocytes of 3W birds is an additional factor that probably favors transient infection. We suggest that lower virus replication is due to a less rapid covalently closed circular DNA amplification, leading to lower viremias and a slower spread of infection in the liver, and that the slower spread of infection in 3W ducks makes the infection more sensitive to interruption by the host immune responses.

ABSTRACT Hepatitis B virus (HBV) frequently causes transient infections in adults but chronic infections in infants. The basis of these age-related outcomes is not known. Infection of ducks with duck hepatitis B virus (DHBV) displays a similar dependence of outcome on the age of the host at the time of infection. In this study we compared the infection of ducks at 3 days and 3 weeks of age. We found that the efficiency of infection of hepatocytes by virus in the inoculum was similar between the two age groups but that spread of the infection throughout the liver was severely inhibited in the 3-week-old-old ducks, while a rapid spread of the infection was observed in 3-day-old ducklings. Inhibition of virus spread was accompanied by the appearance in the serum of virus neutralizing activity, as assayed by blocking of infection of primary hepatocyte cultures. Neutralizing activity appeared as early as 1 or 2 days postinfection and increased during the next 2 weeks. Depletion of immunoglobulins from serum eliminated the neutralizing activity. The specific depletion of IgM indicated that IgM appeared as the dominant fraction of neutralizing antibody in the first 2 days postinfection, but declined from day 3 on while IgG antibody rose. We conclude that excess neutralizing antibody arising rapidly in birds inoculated at 3 weeks of age but not in newly hatched ducks prevented secondary cycles of infection, resulting in a limited infection in the liver and contributing to the eventual transient outcome of the infection.

Integrated hepadnaviral DNA in livers and tumors of chronic hepatitis B patients has been reported for many years. In this study, we investigated whether hepatitis B virus DNA integration occurs preferentially at sites of cell DNA damage. A single I-SceI homing endonuclease recognition site was introduced into the DNA of the chicken hepatoma cell line LMH by stable DNA transfection, and double-strand breaks were induced by transient expression of I-SceI after transfection of an I-SceI expression vector. Alteration of the target cleavage site by imprecise nonhomologous end joining occurred at a frequency of approximately 10(-3) per transfected cell. When replication of an avian hepadnavirus, duck hepatitis B virus, occurred at the time of double-strand break repair, we observed integration of viral DNA at the site of the break with a frequency of approximately 10(-4) per transfected cell. Integration depended on the production of viral double-stranded linear DNA and the expression of I-SceI, and integrated DNA was stable through at least 17 cell divisions. Integration appeared to occur through nonhomologous end joining between the viral linear DNA ends and the I-SceI-induced break, because small deletions or insertions were observed at the sites of end joining. The results suggest that integration of hepadnaviral DNA in infected livers occurs at sites of DNA damage and may indicate the presence of more widespread genetic changes beyond that caused by viral DNA integration itself [corrected].

We determined the frequency of integrated viral DNA in the livers of three woodchucks chronically infected with the woodchuck hepatitis virus before and during 30 weeks of therapy with the nucleoside analog L-FMAU [1-(2-fluoro-5-methyl-beta, L-arabinofuranosyl)uracil, clevudine]. We found that although viral covalently closed circular DNA declined 20- to 100-fold, integrated viral DNA showed no discernable decrease over the course of treatment. Thus, chemotherapeutic clearance of covalently closed circular DNA did not involve the replacement of the infected hepatocyte population with uninfected progenitors, but rather, uninfected hepatocytes in the treated liver were derived from the infected hepatocyte population. The frequency of integrated DNA in chronically infected woodchucks was found to be 1 or 2 orders of magnitude higher than that in transiently infected woodchucks, implying that integration and other genomic damage accumulate over the duration of infection. Our results indicate that genetic changes from this damage remain in the liver even while virus infection is cleared and argue for early antiviral intervention in chronic hepatitis.

Hepatitis B virus (hepadnavirus) infections are maintained by the presence of a small and regulated number of episomal viral genomes [covalently closed circular DNA (cccDNA)] in the nuclei of infected cells. Although a number of studies have measured the mean copy number of cccDNA molecules in hepadnaviral-infected cells, the distribution of individual copy numbers have not been reported. Using a PCR-based assay, we examined the number of cccDNA molecules of the duck hepatitis B virus in single nuclei isolated from the liver of a chronically infected duck over the course of 131 days of infection. Nuclei were isolated from frozen serial biopsies and individually deposited into PCR microplates by flow sorting. Each nucleus was assayed by nested PCR for cccDNA and for cellular IFN-alpha genes as an internal control. We found that 90% of the nuclei assayed contained between 1 and 17 cccDNA molecules, with the remaining 10% containing more (90% confidence), and that differences in the mean number of copies and distribution of copy numbers occurred within the same animal at different times postinfection. Overall, the data suggest (i) that the number of cccDNA molecules per cell may fluctuate over time, and (ii) that, according to these fluctuations, a substantial fraction of cells may contain only one or a few copies. We infer from the results that infected hepatocytes express virus at different levels and that during cell division it is possible to segregate cells containing no cccDNA.

We estimated the amount of hepatocyte turnover in the livers of three woodchucks undergoing clearance of a transient woodchuck hepatitis infection by determining the fate of integrated viral DNA as a genetic marker of the infected cell population. Integrated viral DNA was found to persist in liver tissue from recovered animals at essentially undiminished levels of 1 viral genome per 1,000-3,000 liver cells, suggesting that the hepatocytes in the recovered liver were derived primarily from the infected cell population. We determined the single and multicopy distribution of distinct viral cell junctions isolated from small pieces of liver after clearance of the infection to determine the cumulative amount of hepatocyte proliferation that had occurred during recovery. We estimated that proliferation was equivalent to a minimum of 0.7-1 complete random turnovers of the hepatocyte population of the liver. Our results indicated that during resolution of the transient infections a large fraction of the infected hepatocyte population was killed and replaced by hepatocyte cell division.

Within its host cell, a complex coupling of transcription, translation, genome replication, assembly, and virus release processes determines the growth rate of a virus. Mathematical models that account for these processes can provide insights into the understanding as to how the overall growth cycle depends on its constituent reactions. Deterministic models based on ordinary differential equations can capture essential relationships among virus constituents. However, an infection may be initiated by a single virus particle that delivers its genome, a single molecule of DNA or RNA, to its host cell. Under such conditions, a stochastic model that allows for inherent fluctuations in the levels of viral constituents may yield qualitatively different behavior. To compare modeling approaches, we developed a simple model of the intracellular kinetics of a generic virus, which could be implemented deterministically or stochastically. The model accounted for reactions that synthesized and depleted viral nucleic acids and structural proteins. Linear stability analysis of the deterministic model showed the existence of two nodes, one stable and one unstable. Individual stochastic simulation runs could access and remain at the unstable node. In addition, deterministic and averaged stochastic simulations yielded different transient kinetics and different steady-state levels of viral components, particularly for low multiplicities of infection (MOI), where few virus particles initiate the infection. Furthermore, a bimodal population distribution of viral components was observed for low MOI stochastic simulations. The existence of a low-level infected subpopulation of cells, which could act as a viral reservoir, suggested a potential mechanism of viral persistence.

ABSTRACT In this study, we measured the frequency of revertants of a cytopathic strain of the duck hepatitis B virus that bears a single nucleotide substitution in the pre-S envelope protein open reading frame, resulting in the amino acid substitution G133E. Cytopathic virus mixed with known amounts of a genetically marked wild-type virus was injected into ducklings. Virus outgrowth was accompanied by a coselection of wild-type and spontaneous revertants during recovery of the ducklings from the acute liver injury caused by death of the G133E-infected cells. The frequency of individual revertants in the selected noncytopathic virus population was estimated by determining the ratio of each revertant to the wild-type virus. Spontaneous revertants were found to be present at frequencies of 1 × 10 −5 to 6 × 10 −5 per G133E genome inoculated. A mathematical model was used to estimate that the mutation rate was 0.8 × 10 −5 to 4.5 × 10 −5 per nucleotide per generation.

The dynamic state of infection of 11 ducks with the duck hepatitis B virus was investigated. Chronic infections were established in newly hatched ducklings by inoculation with a mixture of wild-type virus and a mutant virus with a partial replication defect. As expected, the wild-type virus was rapidly enriched in the virus population during the spread of infection. Enrichment thereafter was correlated with normal growth of the liver, with the average mutant-to-wild-type ratio stabilizing for at least 2 months beyond the time at which the liver mass stabilized. Using experimentally determined growth rates for the mutant and wild-type viruses, we estimated that after the spread of infection, competition between the two virus strains was limited by the amount of replication required to infect new hepatocytes in the growing livers. The results suggest that, in a chronically infected liver, the selection of variants with a replication rate advantage is inefficient and that the emergence of such variants would depend on induced liver cell turnover, such as that occurring during chronic hepatitis.

DNA of the avian hepadnavirus, duck hepatitis B virus, was found to be integrated at low abundance into the cellular DNA extracted from the livers of infected ducklings. The frequency of integration was estimated to be at least one viral genome per 10(3) to 10(4) cells by 6 days postinfection. The structures of virus-cell junctions determined by sequencing were compared with those of virus-virus junctions formed by nonhomologous recombination between the ends of linear viral DNA forms. This comparison allowed us to conclude that linear viral DNA was the preferential form used as an integration substrate. Potential factors promoting viral DNA integration during chronic infection are discussed.

ABSTRACT Evidence is presented that the previously cloned type I duck interferon (DuIFN) cDNA encodes a homologue of mammalian interferon-alpha (IFN-α). Recombinant DuIFN-α was used to study the inhibition of duck hepatitis B virus (DHBV) replication in primary hepatocytes in order to determine the IFN-sensitive steps of the virus replication cycle. IFN-treated cells accumulated two- to threefold-lower amounts of viral RNA transcripts early during infection, when IFN was added before virus. This reduction was not due to inhibition of virus entry since initial covalently closed circular DNA levels were not decreased in IFN-treated cells. Interestingly, the inhibitory effect of IFN on viral RNA levels was not observed in cells infected with a mutant DHBV that fails to synthesize core protein, suggesting that an uncharacterized core protein-mediated enhancing effect is blocked by IFN. When IFN was added at 4 days postinfection, encapsidated viral RNA pregenomes disappeared from infected cells within 3 days. This depletion was not simply due to conversion of pregenomes to DNA since depletion was not blocked by phosphonoformic acid, an inhibitor of the viral reverse transcriptase. The intracellular concentration of intact nucleocapsids was reduced, suggesting that in the presence of IFN pregenome-containing capsids were selectively depleted in hepatocytes. Thus, two steps in DHBV replication that involve the viral core protein were inhibited by DuIFN-α.

A variant avian hepadnavirus that has been shown to destroy hepatocytes in vitro was found to be cytopathic in vivo. A single amino acid change of glycine to glutamic acid at position 133 (G133E) in the preS protein of duck hepatitis B virus (DHBV) caused an increase in the intranuclear pool of viral covalently closed circular DNA (cccDNA), resulting in a transient elevation of viral replication and eventual hepatocyte destruction. In vivo viral infection with the G133E virus was compared with infection with wild-type virus over a 72-day period. Birds were inoculated with virus at day 2 post-hatch to ensure a high percentage of infected hepatocytes and potential persistence of virus. Birds infected with the G133E virus had increased periportal cellular proliferation and numerous lysed apoptotic hepatocytes following 100% infection of hepatocytes. The liver damage within G133E virus-infected birds subsided over time, resulting in mild chronic hepatitis that was similar to that observed within wild-type virus-infected birds. The subsidence of liver damage in G133E virus-infected birds coincided with a reduction of viral cccDNA to wild-type virus levels in the liver. Our study indicates that maintenance of wild-type levels of viral cccDNA promotes persistence of virus infection by establishing a noncytopathic infection.

ABSTRACT A precore-deficient mutant of duck hepatitis B virus (DHBV) produced by site-directed mutagenesis was tested for its ability to compete with wild-type virus in a mixed infection of 3-day-old ducklings. The mutation was shown to produce a cis-acting defect, resulting in a replication rate that was about one-half that of wild-type virus. Accordingly, wild-type virus was rapidly selected during the spread of infection. During the chronic phase of the infection, however, two selection patterns were seen. In 4 of 10 ducks, the wild-type virus slowly replaced the precore mutant. In another four ducks, the precore mutant virus slowly replaced the wild-type virus. In the remaining two ducklings, ratios of wild-type and precore mutant virus fluctuated, with wild-type virus slowly predominating. The replacement of wild-type virus was not due to the emergence of a rapidly replicating variant of the precore mutant, since genomes cloned from the infected ducks retained their original replication defect. Replacement of wild-type virus, however, correlated with elevated anti-core antibody titers, which continued to increase with time. The selection of a precore-negative strain of DHBV may be analogous to the selection for precore mutants of HBV during chronic hepatitis in humans.

Several examples of human hepatitis B virus strains with enhanced replication in vitro have been described. To understand whether this characteristic could be a cause of liver disease, we have studied a variant of the closely related duck hepatitis B virus (DHBV) that had enhanced levels of cccDNA accumulation, previously shown to be cytopathic in vitro, as a model for the pathogenesis of analogous viruses in humans. In vivo liver damage caused by this variant (G133E) occurred only during the first 2 weeks p.i., after which time cccDNA levels and liver histology returned to near normal despite continued virus replication. To determine whether recovery was due to the emergence of noncytopathic revertant, we tested whether wild-type virus would have a selective advantage in competition with the cytopathic mutant in a fully infected liver. In a mixed infection of ducklings with G133E and a small amount of wild-type virus, the wild-type virus was detected as the predominant genotype after recovery of normal liver histology. Two candidate revertant viral genomes were cloned directly from the serum virus of G133E-infected birds after recovery and tested for (i) control of cccDNA levels in primary hepatocyte cultures and (ii) their ability to compete with wild-type virus in a mixed infection. At least one noncytopathic revertant was identified by these two criteria. The results support the conclusion that the recovery from liver damage in G133E-infected ducklings was due to the emergence of spontaneous noncytopathic revertants rather than to host suppression of virus cytotoxicity. The results indicate that acute liver injury may result from infection with a cytopathic hepadnavirus but that such viruses may be rapidly replaced by noncytopathic variants during persistent infection.

ABSTRACT Double-stranded linear DNA is synthesized as a minor viral DNA species by all hepadnaviruses. In a previous study (W. Yang and J. Summers, J. Virol. 69:4029–4036, 1995) we showed that virus particles containing linear DNA of the duck hepatitis B virus (DHBV) could initiate an infection of primary duck hepatocytes. In cells infected by linear DNA containing viruses the transcriptional template, covalently closed circular DNA, was formed by circularization of linear DNA by nonhomologous recombination between the two ends. This process was shown to result in viral DNA replication through multiple generations of linear DNA intermediates, a process we called illegitimate replication. In this study we showed that viruses containing linear DHBV DNA produced by engineered insertions in the r sequence, which encodes the 5′ end of the pregenome, could infect hepatocytes in vivo, and these hepatocytes proceeded to carry out illegitimate replication. Nonhomologous recombination quickly produced revertants and partial revertants in which all or part of the insertion was deleted. One such partial revertant that replicated primarily through circular DNA intermediates, but which synthesized elevated levels of linear DNA, could be sustained for several days as the predominant genotype in vivo, but this mutant was eventually displaced by variants showing full reversion to legitimate replication and that synthesized normal low levels of linear DNA. Full revertants did not necessarily contain the wild-type r sequence. The results suggest that the linear DNA produced during DHBV infection initiates cycles of illegitimate replication by generating mutants with altered r sequences. Some r sequence mutants carry out a mixture of legitimate and illegitimate replication that can contribute to elevated production of linear DNA in individual cells.

We report the presence of two elements, pet and net, that are required for proper transcription of the duck hepatitis B virus (DHBV). These regions were previously identified by using plasmid clones of the virus in transient expression assays (M. Huang and J. Summers, J. Virol. 68:1564-1572, 1994), In this study, we further analyzed these regions by using in vitro-synthesized circular DHBV DNA monomers to mimic the authentic transcriptional template, We observed that pet was required for pregenome transcription from circular viral monomers, and in the absence of pet-dependent transcription, expression of the viral envelope genes was increased, We found that deletion of net in circularized DNA monomers led to the production of abnormally long transcripts due to a failure to form 3' ends during transcription, In addition, we report the presence of a net-like region in the mammalian hepadnavirus woodchuck hepatitis virus, These results are consistent with a model that net is a region involved in transcription termination and that in DHBV, pet is required for transcription complexes to read through this region during the first pass through net.