Increased Fidelity Reduces Poliovirus Fitness and Virulence under Selective Pressure in Mice

Julie K Pfeiffer
Department of Microbiology and Immunology
Stanford University School of Medicine
StanfordCaliforniaUnited States of America

Karla Kirkegaard
Department of Microbiology and Immunology
Stanford University School of Medicine
StanfordCaliforniaUnited States of America

Increased Fidelity Reduces Poliovirus Fitness and Virulence under Selective Pressure in Mice

RNA viruses have high error rates, and the resulting quasispecies may aid survival of the virus population in the presence of selective pressure. Therefore, it has been theorized that RNA viruses require high error rates for survival, and that a virus with high fidelity would be less able to cope in complex environments. We previously isolated and characterized poliovirus with a mutation in the viral polymerase, 3D-G64S, which confers resistance to mutagenic nucleotide analogs via increased fidelity. The 3D-G64S virus was less pathogenic than wild-type virus in poliovirusreceptor transgenic mice, even though only slight growth defects were observed in tissue culture. To determine whether the high-fidelity phenotype of the 3D-G64S virus could decrease its fitness under a defined selective pressure, we compared growth of the 3D-G64S virus and 3D wild-type virus in the context of a revertible attenuating point mutation, 2C-F28S. Even with a 10-fold input advantage, the 3D-G64S virus was unable to compete with 3D wild-type virus in the context of the revertible attenuating mutation; however, in the context of a non-revertible version of the 2C-F28S attenuating mutation, 3D-G64S virus matched the replication of 3D wild-type virus. Therefore, the 3D-G64S high-fidelity phenotype reduced viral fitness under a defined selective pressure, making it likely that the reduced spread in murine tissue could be caused by the increased fidelity of the viral polymerase. Citation: Pfeiffer JK, Kirkegaard K (2005) Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice. PLoS Pathog 1(2): e11.

Introduction

RNA viruses have the highest replicative error rates in nature, with approximately one mistake made per 1,000-100,000 nucleotides copied [1][2][3]. As a result, each viral genome is likely to differ from every other virus by one or more point mutations. The complex populations thus generated, called quasispecies, have been hypothesized to be important for survival of the population as a whole in the presence of selective pressure, under which a few viruses that contain beneficial mutations would survive and act as founders for the next generation [1,4]. For example, viruses with mutations that confer resistance to neutralizing antibodies would benefit the virus population as a whole under selective pressure from the host immune response. However, the vast majority of errors made during replication are deleterious, resulting in debilitation of a high percentage of the population. Therefore, RNA viruses live on the edge of ''error catastrophe,'' where the cost of deleterious mutations is in dynamic balance with the benefit of adaptive mutations [1,5,6].

A promising new class of antiviral drugs, RNA virus mutagens, takes advantage of the cost of error catastrophe. Ribavirin is a nucleoside analog mutagen, which is incorporated into the viral genome during replication and increases error frequency by promiscuous base-pairing [3]. Ribavirin debilitates the viral population over several rounds of replication by causing accumulation of deleterious mutations and error catastrophe [5]. Previously, we isolated a poliovirus variant able to survive serial passage in ribavirin, and mapped this phenotype to a single mutation in the viral polymerase, 3D-G64S [7]. The mechanism of ribavirin resistance in 3D-G64S mutant poliovirus was shown to be due to increased fidelity of RNA replication by showing that it has lower reversion frequency at the resistance mutation for the antiviral compound guanidine [7], confers resistance to other mutagens as well [7], by direct sequencing of viral genomes [8], and by demonstration that the purified 3D-G64S poliovirus displays increased fidelity in solution that correlates in a delayed conformational change before phosphoryl transfer compared to the wild-type enzyme [8].

If a viral quasispecies is important to the survival either of individual genomes or of the population during growth under standard laboratory conditions, the higher fidelity of 3D-G64S virus should result in an attenuated phenotype. Therefore, we wondered if the 3D-G64S virus would have growth defects in tissue culture, a relatively simple environment, or in mice, a complex environment with many selective pressures. Laboratory strains of mice are not susceptible to poliovirus infection; however, mice transgenic for the human poliovirus receptor, CD155, are susceptible to injected poliovirus [9][10][11]. When poliovirus receptor (PVR) mice are given poliovirus by intramuscular (IM) injection in the leg, animals develop paralysis and eventually die of paralytic disease. Poliovirus inoculated by IM injection is thought to traffic up the sciatic nerve, to the spinal cord, and then to the brain [12,13], during which many different environments are likely to play a role in its propagation.

In this study, we found that 3D-G64S poliovirus is less pathogenic than wild-type virus in mice, but displays little growth defect in tissue culture. This attenuation in mice was at least partially due to the increased fidelity of the 3D-G64S virus in the context of a defined selected pressure, since it was unable to compete with 3D wild-type virus in the context of a revertible attenuating point mutation. Therefore, we argue that, in the face of the myriad selective pressures encountered by poliovirus during murine infection, the decreased variability in the population generated by the relatively highfidelity 3D-G64S polymerase may be a distinct disadvantage for the growth and spread of the mutant virus.

Results

3D-G64S Virus Is Less Pathogenic than Wild-Type Virus in Mice

To test whether the pathogenicity of high-fidelity 3D-G64S virus was comparable to that of wild-type virus in infected animals, mice that express the human poliovirus receptor [9] were inoculated by IM injection with 5 3 10 6 PFU (plaqueforming units), an amount slightly over the LD50 for these animals [9]. Animals infected in this way develop symptoms similar to poliomyelitis in humans [10]. First, animals display lethargy, then weakness and paralysis in the inoculated leg, then paralysis of both legs, followed quickly by death. For this study, we monitored the time required for the first symptoms, weakness and paralysis of the inoculated limb, to develop. Figure 1 shows time courses for 48 mice inoculated with either wild-type or 3D-G64S virus. The 3D-G64S-inoculated animals developed disease significantly more slowly than wild-type infected animals, and fewer animals succumbed to disease ( Figure 1). Therefore, the 3D-G64S virus is less pathogenic than wild-type virus in mice.

Competition between 3D-G64S and Wild-Type Virus in Mice

To test whether the decreased pathogenicity of the 3D-G64S virus could be observed in mixed infections, we infected PVR mice with mixtures of wild-type and 3D-G64S virus. A 1:2 ratio of wild-type:3D-G64S virus was chosen because, as will be shown in subsequent figures, this ratio was required to achieve equal RNA yields in mixed infections of HeLa cells. A total of 2 3 10 7 total PFU of mixed virus were given as IM injections and mice were sacrificed at the first sign of disease, usually 3 or 4 d post-infection. To monitor the relative accumulation of the mutant and wild-type RNAs of mixed infections, we took advantage of the presence of an additional BstBI restriction enzyme site engineered at the position of the 3D-G64S mutation. As shown in Figure 2A, BstBI digestion of end-labeled RT-PCR reaction products from mixtures of wild-type and 3D-G64S RNAs is expected to produce products of different sizes. For the sets of primers used here, a 558-basepair (bp) fragment is produced from RT-PCR analysis of wild-type RNA, and a 396-bp product from 3D-G64S RNA. Although this assay was not designed to quantify the total amounts of these viral RNAs, it should accurately determine their relative proportions within a population. Inoculated leg muscles and brains were harvested from mice infected with a mixture of wild-type and 3D-G64S viruses, RNA was extracted, and the products of reverse transcription, PCR amplification, end-labeling and BstBI digestion were quantified. As shown in Figure 2B and quantified in Figure 2E, both wild-type and 3D-G64S viruses were able to replicate within the inoculated muscle tissue. However, when products from brains were analyzed ( Figures  2C and 2F), a distribution of outcomes was observed: in many mice (69%), only the wild-type band was present (e.g., mice 1, 2, 4, 6); in some mice (22%), the 3D-G64S band was predominately or exclusively present (e.g., mouse 5), and only rarely (9%) were both bands present (e.g., mouse 7). These results support two conclusions; first, 3D-G64S virus has a defect in spreading to the brain following IM infection, and second, because usually only one of the two viruses was found in the brain, there is a bottleneck between the injection site and the brain. This bottleneck to poliovirus spread has

Synopsis

RNA viruses have the highest error rates in nature, resulting in the likelihood that each virus differs from other viruses in the population by one or more mutations. The consequence of this ''infidelity'' is that the viral population as a whole, under selective pressure from the immune system or antiviral drugs, may benefit from adaptive changes in a subset of its members. Therefore, it has been theorized that RNA viruses need high error rates to survive in complex environments. We tested this hypothesis using a drug-resistant poliovirus that contains a mutation in its polymerase that reduces errors during replication. We found that this high-fidelity mutant virus has reduced growth in mice, a complex environment where mutations may be required for growth and spread within the infected animal. At least part of this attenuation is likely due to the high fidelity of this mutant virus, since it was unable to compete with the low-fidelity version of the virus in the context of a defined selective pressure. Therefore, it is likely that mutations do benefit viral populations, especially in complex environments such as an infected animal or human.

been observed previously in both humans [14,15] and in mice [15] (J. K. Pfeiffer, unpublished data).

To ensure that both viruses were able to replicate and be detected in the brain at the same time, intracerebral (IC) inoculations were performed using a 1:1 input ratio of wildtype:3D-G64S virus. Samples from the brains of these mice ( Figures 2D and 2G) showed that both viruses were able to replicate in the brain when inoculated into this tissue. Therefore, the preferential spread of wild-type virus from the infected muscle to the brain did not result from an inability of 3D-G64S virus to replicate in murine brain tissue.

3D-G64S Displays a Slight Growth Defect in Cultured Cells

The decreased virulence of the high-fidelity 3D-G64S mutant virus ( Figure 1) could result from: (1) an advantage conferred to the wild-type virus by the increased complexity showing BstBI restriction enzyme sites within the coding region for 3D polymerase. Digestion of 1,024-bp 32 P endlabeled (*) RT-PCR products yields a 558-bp labeled band for wild-type and a 396-bp labeled band for 3D-G64S. 6-wk-old mice were given IM injections with a 1/2 mixture of wild-type to 3D-G64S poliovirus (2 3 10 7 PFU total virus per mouse). Tissues were harvested when animals first developed paralysis, between days 3 and 5. PCR products were end-labeled with 32 P during synthesis by inclusion of a radiolabeled downstream primer and products were digested with BstBI before electrophoresis on a denaturing polyacrylamide gel. Products from muscle samples from the IM infections are shown in (B) while products from brain samples of the IM infections are shown in (C). In (D), 2-wk-old mice were given IC injections with a 1/1 mixture of wild-type to 3D-G64S virus, and brain tissue was harvested when mice became lethargic or paralyzed. For (B), (C), and (D), representative gels are shown. In (E), (F), and (G) all mouse data obtained from the types of experiments shown in (B), (C), and (D), respectively, are quantified, and show the total number of mice whose tissues contained predominately wild-type virus, 3D-G64S virus, or both. The total number of mice per condition are as follows: IM-muscle samples, 31 mice; IM-brain samples, 54 mice; IC-brain samples, 10 mice. DOI: 10.1371/journal.ppat.0010011.g002 of its quasispecies population, or (2) a growth disadvantage conferred to the 3D-G64S mutant virus by some other property of the mutant polymerase than its increased fidelity. To monitor the replication of high-fidelity 3D-G64S poliovirus in tissue culture, several kinds of viral growth assays were performed. After infection of HeLa cell monolayers with wild-type and 3D-G64S virus at a multiplicity of infection (MOI) of 10 PFU/cell, the growth curves of singlecycle infections were found to be indistinguishable ( Figure  3A). Furthermore, when wild-type and 3D-G64S virus growth was compared in plaque assays ( Figure 3B), the plaques were of similar size at all temperatures tested, indicating equivalent growth of the viruses for several rounds of replication in HeLa cells.

To test for a defect in RNA accumulation in the 3D-G64S mutant virus, we performed competition assays in HeLa cells with wild-type and G64S mutant virus and monitored the relative proportions of RNA after single-cycle infections. To determine the sensitivity of the competition assay, plasmid DNAs that contained wild-type and 3D-G64S cDNAs were mixed at various ratios and the 3D polymerase coding regions were amplified and end-labeled by PCR. The BstBI-digested products from the wild-type and 3D-G64S genomes ( Figure  2A) displayed equivalent intensities when obtained from a 1:1 mixture of wild-type and 3D-G64S mutant cDNAs ( Figure  3C). At 1:10 input ratios, however, the bands derived from the minority species were barely visible, and disappeared altogether at 1:100 input ratios. Therefore, this assay is capable of detecting both the wild-type and 3D-G64S-derived products, as long as the input amounts are within 10-fold of each other ( Figure 3C). When the relative amounts of RNA accumulation of wild-type and 3D-G64S virus from single and mixed infections of HeLa cell monolayers were monitored, a slight RNA accumulation defect for the 3D-G64S virus was observed. As shown in Figure 3D, a 1:1 input ratio of the two viruses displayed a decreased accumulation of 3D-G64S RNA relative to that of wild-type. Using twice as much 3D-G64S virus in the mixed infection equalized the bands ( Figure 3D). Therefore, although no defect was observed in viral growth assays in HeLa cells, 3D-G64S virus displayed a slight RNA accumulation defect during a single cycle of replication.

Viral growth in HeLa cells, however, may not mirror replication capacity in murine cells, and subtle growth defects may be more apparent in primary cells. Therefore, we compared the growth of wild-type and 3D-G64S viruses in mouse embryo fibroblasts (MEF) derived from PVR mice [9]. In single-cycle growth curves in these primary cells, 3D-G64S (C) Competition assay titration with plasmid DNA. Plasmid DNAs that encode wild-type and 3D-G64S poliovirus were diluted and mixed at the ratios indicated: for example, 1/100 denotes a ratio of 0.1 ng of wild-type viral cDNA to 10 ng of 3D-G64S cDNA. End-labeled PCR products were digested with BstBI and were electrophoresed on denaturing polyacrylamide gels. (D) Competition assay in HeLa cells infected with wild-type and 3D-G64S virus. HeLa cells (2 3 10 6 ) were infected at an MOI of 0.1 or 10 PFU/cell, as indicated, with wild-type virus, 3D-G64S virus, or both, and cells were harvested 5 h after infection. RNA was extracted, subjected to RT-PCR as in (C), and digested with BstBI. Control bands ''uncut'' and ''mix'' were amplified from cDNAs. Products from MOI 0.1 single infections with wildtype, 3D-G64S-and mock-infected cells are shown, as are mixed infections with a 1/1 ratio of wild-type and 3D-G64S virus (MOI of 0.1 PFU/cell or 10 PFU/cell for each virus) and a 1/2 ratio (MOI of 5 and 10 PFU/cell, respectively). Products were quantified by Phosphorimager and the percentage 3D-G64S is shown. DOI: 10.1371/journal.ppat.0010011.g003

October 2005 | Volume 1 | Issue 2 | e11 0105 virus showed slightly reduced accumulation at early time points ( Figure 4A), displaying 2-to 4-fold titer reduction compared to wild-type virus at 3, 4, and 5 h post-infection. Despite this slight growth defect in a single-cycle growth curve, wild-type and 3D-G64S viruses displayed similar plaque sizes in the PVR-MEF ( Figure 4B). To test whether this slight growth defect would prevent the 3D-G64S virus from propagating during multiple passage in the PVR-MEF, RNA from serially passaged mixtures of wild-type and G64S virus was analyzed. PVR-MEF were inoculated with a 1:1 input ratio of mutant:wild-type virus at high MOI (5-30 PFU/cell). As shown in Figure 4C, the 3D-G64S and wild-type product amounts were comparable over three rounds of serial passage, indicating no significant growth defect exists for the mutant virus in PVR-MEF even during multiple rounds of infection. It should be noted that poliovirus replication is 10to 100-fold less efficient in MEF compared with HeLa cells (Figures 3A, 4A and 4B); therefore, MEF represent a stringent environment, where growth defects may be more apparent. The fact that 3D-G64S virus matches the replication of wildtype virus in MEF indicates the mutant virus has only a slight growth defect in cultured cells.

Effect of a Defined Selective Pressure on the Fitness of 3D-G64S Mutant Virus: Characterization of the Selective Pressure

The reduced pathogenesis of the 3D-G64S mutant virus could result, as stated previously, from a growth defect or the quasispecies restriction caused by increased polymerase fidelity. In the latter case, the increased mutation rate in the wild-type virus population would presumably aid in viral spread in the diverse environments encountered during spread through the animal. We do not know what these diverse selection pressures might be; therefore, we chose to exert a defined selective pressure on the viral mixtures of wild-type and G64S mutant virus and inquire whether the high-fidelity phenotype of the 3D-G64S virus became a liability. Specifically, a single amino acid change (F28S) in the coding region for poliovirus protein 2C is known to confer temperature sensitivity to the virus in cell culture [16] (J. K. Pfeiffer, unpublished data). We engineered two versions of the F28S amino acid change: one version that contained a single nucleotide substitution [2C-F28S(1m)], which should be able to revert readily during propagation, and a second version that contained three nucleotide substitutions [2C-F28S(3m)], to reduce the probability of reversion to the wildtype codon ( Figure 5). These two versions of the F28S mutation were cloned into wild-type and 3D-G64S backgrounds and the resulting viruses were plaque-purified and propagated at 32 8C, the permissive temperature. No differences in the rates of growth of viruses that contained the 2C-F28S(1m) and 2C-F28S(3m) mutations were observed during single-cycle infections in cell culture (J. K. Pfeiffer, unpublished data). As shown in Figure 5, the presence of either the single-nucleotide (1m) or the three-nucleotide (3m) 2C-F28S mutation did not change the ability of the 3D-G64S virus to compete with virus that was wild-type at the 3D locus during serial passage in HeLa cells.

To determine the effect of the 2C-F28S(1m) and 2C-F28S(3m) mutations on pathogenesis and growth in mice, and the role of the probability of genetic reversion in these processes, we inoculated PVR mice intramuscularly with the  Figure 6A). The ratio of 1:10 (wild-type: 3D-G64S) at the 3D locus was chosen to enhance the detection of 3D-G64S-containing RNAs, should they compete poorly with virus that was wild-type at the 3D polymerase locus. 2-wk-old mice were inoculated by IM injection with the three mixtures of viruses and neuropathological symptoms were monitored as a function of time. Upon injection of 5 3 10 6 PFU, mice inoculated with virus mixtures that were wild-type at the 2C locus showed symptoms by the third day post-infection, whereas mice inoculated with the 2C-F28S(1m) virus mixture showed a small, but significant, delay in disease onset ( Figure 6A). Even more strikingly, the 2C-F28S(3m) virus mixture was found to cause no disease at the input dose of 5 3 10 6 PFU ( Figure 6A). At higher doses, however, the 2C-F28S(3m) virus mixture caused disease ( Figure 6B). We hypothesized that the 2C-F28S mutation is highly attenuating, as shown by the profoundly reduced pathogenesis of the 2C-F28S(3m) virus mixture. However, because the 2C-F28S(1m) mutations could readily revert to a wild-type sequence, the 2C-F28S(1m) virus mixture caused much more disease than the 2C-F28S(3m) mixture. To test this hypothesis, the viral RNAs present in muscle tissue from diseased mice [wild-type and 2C-F28S(1m) from Figure 6A, and 2C-F28S(3m) from Figure 6B] were amplified and sequenced in the region of the 2C mutations. Portions of the resulting chromatograms, as well as sequences obtained from RT-PCR products of the viral RNAs in the input pools, are shown in Figure 6C. As predicted, the sequences near the site of the mutations at the 2C locus from the wild-type pool, and the 2C-F28S(3m) pool did not change during passage in the mouse. However, virus isolated from mice infected with the 2C-F28S(1m) pool showed a high frequency of reversion to wild-type sequence at the F28S(1m) codon ( Figure 6C). Therefore, the increased pathogenesis of the F28S(1m) virus pool, relative to that of the F28S(3m) virus pool, was accompanied by direct reversion of the F28S(1m) mutation, implying that there was strong selective pressure for wildtype sequence at this codon.

Effect of a Defined Selective Pressure on the Fitness of 3D-G64S Mutant Virus: Growth in Mouse Tissue

To ask whether the presence of the strong selective pressure to revert the 2C-F28C mutation would decrease the competitiveness of 3D-G64S virus, we analyzed the relative ratios of 3D-G64S and wild-type RNA sequence at the 3D locus by analysis of muscle samples from many of the mice shown in Figure 6. For the mixture of viruses that were wild-type at the 2C locus ( Figure 7A), the 3D-G64S genomes were more highly represented than genomes that were wildtype at the 3D locus, which was an expected result considering the 10-fold excess of the 3D-G64S virus in the inoculum. However, in the context of the ''revertible'' attenuating mutation, 2C-F28S(1m), the 3D-G64S products were poorly represented in the resulting population ( Figure  7B). However, in the presence of the ''non-revertible'' 2C-F28S(3m) mutations, the 3D-G64S products were once again well-represented in the population ( Figure 7C). These results, quantified in Figure 7D, argue that the high fidelity of the 3D-G64S virus decreases its fitness in the presence of a defined selective pressure, if errors introduced by the wild-type polymerase can bring about escape from that pressure.

Discussion

It has been theorized that RNA viruses need high error rates to survive in complex environments [1]. With the isolation of a high-fidelity poliovirus, we were in a unique position to address this hypothesis.

In tissue culture, we found a slight RNA accumulation defect of 3D-G64S poliovirus in HeLa cells (Figure 3), which may result from the decreased rate of nucleotide incorporation observed in reactions with purified 3D-G64S polymerase [8]. In murine embryo fibroblasts derived from PVRexpressing mice, a slight growth defect was also observed in single-cycle infections ( Figure 4A). Nevertheless, in both HeLa cells and PVR-MEF, the 3D-G64S could compete with wild-type virus over several rounds of serial passage (Figures 4  and 5). Our results differ from those of Arnold et al, where population sequencing suggested the 3D-G64S virus could not compete with wild-type virus even during one round of competition passage [8]. We do not know the basis for this discrepancy, but it could be due to differences in the virus clones used, the method of detection, or the cell lines.

In mouse infections, the 3D-G64S virus was less pathogenic than wild-type virus, as measured by the time of symptom onset and the number of animals acquiring disease (Figure 1). When mice were inoculated with a mixture of the wild-type and 3D-G64S viruses, both were able to replicate in muscle and both were capable of spreading to the brain, although 3D-G64S virus spread to the brain less often than wild-type virus. Interestingly, we found that generally, only one of the two input viruses was present in the brain of infected animals. This so-called poliovirus ''bottleneck effect'' has been observed before, both in mice and in humans [14,15]. Other pathogens also encounter bottlenecks during spread within a host. The plant RNA virus Cucumber mosaic virus undergoes striking bottlenecks during spread within an infected plant [17], and Salmonella strains and Pneumocystis carnii experience bottlenecks during spread in rodents [18][19][20]. We will describe the nature of the poliovirus bottleneck effect in more detail in the future. The virulence and growth defects of 3D-G64S poliovirus in mice could be due to its enhanced fidelity or to the slight growth defect documented here. To determine whether enhanced fidelity could play a role in the fitness of poliovirus, we competed 3D-G64S virus against virus that was wild-type at the 3D locus in the context of a defined selective pressure, a revertible attenuating point mutation in the viral 2C coding region. Indeed, the 3D-G64S virus was unable to compete with virus that was wild-type at the 3D locus in the presence of a revertible version of the 2C mutation. This did not result from a growth defect of the 3D-G64S virus, because, in the absence of the 2C mutation, or in the presence of a nonrevertible version of the 2C mutation, the 3D-G64S-containing virus could compete well with virus that was wild-type at the 3D polymerase locus (Figure 7).

The results presented here argue that a high-fidelity poliovirus variant that contains the 3D-G64S mutation in the coding region for its RNA-dependent RNA polymerase is less able to revert attenuating point mutations. Therefore, in the context of known, harmful, mutations, a virus that is wildtype at the 3D polymerase locus is more fit than the higherfidelity 3D-G64S virus. Most of the mutations made during viral growth are likely to be deleterious, and it is possible that reversion of attenuating mutations is important for survival of RNA viruses. However, why would it not be advantageous for a virus to encode a higher-fidelity polymerase and simply not to make so many errors in the first place?

Genetic change can be very rapid in the presence of a lowfidelity polymerase ( Figure 6C). In a complex environment such as within a mouse, the ability to generate a quasispecies may allow virus populations to respond to the different environments they encounter during spread between tissues and in reaction to changing pressure of the host immune response. In monkeys infected with a vaccine strain of poliovirus, distinct mutations were observed to occur within 2 d post-infection, only to revert to the inoculated sequence later in the course of disease [21]. Furthermore, HIV is known to change its cell tropism from macrophage-tropic to T celltropic, during spread in many infected individuals [22][23]. It would be interesting to determine whether the pathogenesis of HIV is reduced by increasing the fidelity of its replicative machinery. We have shown that, in the presence of a defined selective pressure, the increased fidelity of the 3D-G64S mutant virus decreases its fitness. Given the slight growth defect of the 3D-G64S mutant virus, we further suggest that, the decreased virulence of high-fidelity 3D-G64S mutant poliovirus in the mouse is due, at least in part, to its relative inability to generate variants suitable for the variety of selection pressures faced during the normal course of infection of a complex organism.

Materials and Methods

Cells, mice, and viruses. HeLa cells were grown in Dulbecco's modified Eagle's medium, supplemented with 10% calf serum [24]. PVR mice expressing the human poliovirus receptor (CD155) from a b-actin promoter were obtained from R. Andino (University of California at San Francisco) [9]. Primary MEF were harvested from PVR mice. Day 14-18 embryos were harvested, washed in phosphatebuffered saline, the heads and viscera were removed, and the body was minced with a sterile razor blade. The slurry was trypsinized and homogenized with a pipet for 10 min and then plated in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum. Experiments with MEF were performed between passages two and five (see below). Mahoney serotype 1 poliovirus was grown from a single plaque generated by transfection of the viral cDNA clone [25]. Individual plaques were grown into high-titer virus stocks by infecting monolayers of HeLa cells at 37 8C. Plasmids that encoded cDNAs for temperature-sensitive mutant viruses were transfected at 32 8C, and small plaques were picked 4 d later. In all cases, care was taken to ensure that the specific infectivities were comparable to wild-type plasmid cDNAs transfected at the same time. Multiple individual plaques were chosen from the transfections and propagated for three cycles of infection at 32 8C to generate high-titer stocks. The phenotypes of these stocks were then compared to those of the original plaque isolates to ensure that no additional mutations were acquired during propagation.

Plasmid construction. The 3D-G64S mutation in the viral 3Dcoding region was introduced by site-specific mutagenesis, which added a BstBI restriction enzyme site (sense primer: 59ATTTTCTCCAAGTACGTTTCGAACAAAATTACTGAAGTG 39). This BstBI-marked 3D-G64S derivative was engineered to contain three nucleotide substitutions (GGT to TCG) to minimize reversion at this site. A mutation in the 2C coding region, F28S, which confers temperature sensitivity [16], was introduced into both the 3D-G64S and wild-type poliovirus clones in two forms: one with nucleotide substitution [TTC to TCC, termed 2C-F28S(1m)] and the other with three nucleotide substitutions [TTC to AGT, termed 2C-F28S(3m)]. In each case, the entire PCR-generated region was confirmed by sequencing.

Tissue culture infections. For single-cycle viral growth curves, 2 3 10 6 HeLa cells or 2 3 10 5 PVR-MEF were infected at 37 8C at an MOI of 10 PFU/cell, harvested at various times post-infection, and lysed by repeated freeze-thawing. Cell-associated virus was quantified by plaque assay on HeLa cell monolayers [7]. Each growth curve was performed at least twice and results were similar in all replicates. For competition experiments, as shown in Figure 3D, 2310 6 HeLa cells were infected at an MOI of 0.1 or 10 PFU/cell with wild-type virus, 3D-G64S virus, or both viruses, at varying ratios for 5 h at 37 8C. Cells were harvested and RNA was extracted using TRIZOL (Invitrogen, Carlsbad, California, United States) according to the manufacturer's instructions. For serial passage competition experiments shown in Figure 5, 2310 6 HeLa cells were infected at 32 8C with a 1:1 mixture of wild-type and 3D-G64S viruses at low MOI (0.1 PFU/cell). At 12 h post-infection, cell-associated virus and RNA were harvested (as above), and the cycle was repeated three to five times, titering the virus each time to determine input for the next cycle. Serial passage experiments in PVR-MEF ( Figure 4C) were performed as above, except that 2 3 10 6 MEF were infected at a high MOI (5-30 PFU/cell) at 37 8C, and cell-associated virus and RNA were harvested at 6 h post-infection.

Mouse infections and tissue processing. All animal work was performed according to protocols approved by the Stanford University Administrative Panel on Laboratory Animal Care. For IM inoculations, 50 ll of virus was injected into the thigh muscle of the right hind leg of mice that were 2-10 wk old, as indicated, using a 28-gauge needle. Before IC inoculations, 2-wk old animals were anesthetized by intraperitoneal injection with 11 ll of a solution containing 2.5% avertin/g animal weight. Once animals were unconscious, 20 ll of virus stock was injected into the cerebrum through the right temple. Mice were observed for symptoms twice per day, and were euthanized at the first sign of disease, weakness in the inoculated limb, or, for IC-inoculated mice, ruffled fur and general lethargy. Tissues, including the inoculated leg muscle, brain, or both, were harvested and frozen at À80 8C until studied. Tissues were ground into a fine powder under liquid nitrogen using a mortar and pestle. RNA was extracted from approximately 300 mg of total tissue using TRIZOL (Invitrogen) according to the supplied protocol.

Reverse transcription-PCR digestion assay. RNA was reverse transcribed using 5-10 lg of RNA per cDNA synthesis reaction using Superscript II reverse transcriptase (Invitrogen) and reverse primer 59ATAGGTTCCCAGAA GCCATTCTC39 for the 3D polymerasecoding region or 59CCATGTCGAACGCAGAGCGCCTGG39 for the 2C-coding region. Twenty percent of the reverse transcription r e a c t i o n w a s P C R a m p l i fi e d u s i n g 5 9C C G C A T T T -G G A A G T G G A T C T A C T C A G C A G 3 9 a n d 5 9C C A T G T C G -AACGCAGAGCGCCTGG39 primers for the 2C-coding region and primers 59GCTTCACCTGGTGAAAGCATTGTGATCG39 and 59ATAGGTTCCCAGAA GCCATTCTC39 primers for the 3D-coding region. The 3D reverse primer was end-labeled using T4 polynucleotide kinase (New England Biolabs, Beverly, Massachusetts, United States), and 125 lCi [c-32 P] ATP (Perkin-Elmer, Boston, Massachusetts, United States), and was used at a 1:5 ratio with the unlabeled 3D reverse primer in PCR reactions. The PCR reactions contained 1 unit Taq (Qiagen, Valencia, California, United States) and were done according to the supplier protocol, using a buffer that contained 50 lM potassium glutamate, 20 lM potassium HEPES (pH 8.4), 3 lM MgCl 2 and 0.1 mg/ml BSA. The PCR products were digested with BstBI (New England Biolabs) at 65 8C for 3-16 h, and products were ethanol-precipitated in the presence of glycogen (Invitrogen). Pellets were resuspended in denaturing gel loading dye (80% formamide, 10 mM EDTA, 0.025% bromophenol blue and xylene cyanol, 0.1% SDS), heated to 80 8C for 2 min, and loaded on gels that contained 7% polyacrylamide and 8M urea. Gels were dried under vacuum on DE81 paper (Whatman, Florham Park, New Jersey, United States) and exposed to PhosphorImager screens. Sequence analysis. The cDNAs products from viral RNAs purified using the QiaAmp kit (Qiagen) or from cDNA-encoding plasmids were PCR-amplified for sequence analysis at the F28S locus. The PCR reactions were performed as above, except that no radiolabel was added. Products were run on 1% agarose gels and products were excised, purified using the Qiaex II kit (Qiagen), and sequenced commercially (Sequetech, Mountain View, California, United States).

Supporting Information

Accession Number

The Genbank (http://www.ncbi.nlm.nih.gov/entrez) accession number for serotype 1 poliovirus is NC002058.

Figure 1 .
1Pathogenesis of Wild-Type and 3D-G64S Viruses in PVR Mice PVR mice were given IM injections with 5 3 10 6 PFU of either the wildtype or 3D-G64S viruses and were monitored for symptoms of disease.

Figure 2 .
2Viral Competition Assay in Poliovirus Receptor-Expressing Mice (A) Diagram of poliovirus genome

Figure 3 .
3Growth of Wild-Type and 3D-G64S Poliovirus in HeLa Cells (A) Single-cycle growth curve. 2 3 10 6 HeLa cells were infected at an MOI of 10 PFU/cell with either wild-type or 3D-G64S viruses at 37 8C. Cellassociated virus was harvested at indicated times and titered by plaque assay. (B) Plaque assays. Wild-type or 3D-G64S poliovirus was plated on HeLa cell monolayers and incubated under an agar overlay for 48 h at indicated temperatures before staining the cells with crystal violet.

Figure 4 .
4Growth of Wild-Type and 3D-G64S Poliovirus in Primary PVR-MEF (A) Single-cycle growth curve. 2 3 10 5 PVR-MEF were infected at an MOI of 10 PFU/cell with either wild-type or 3D-G64S viruses at 37 8C. Cellassociated virus was harvested at indicated times and titered by plaque assay. (B) Plaque assays. Wild-type or 3D-G64S poliovirus was plated on HeLa or PVR-MEF monolayers and incubated under an agar overlay for 72 h at 37 8C before staining the cells with crystal violet. The amount of virus plated, according to HeLa titration, is shown underneath as ''input.'' (C) Serial passage competition assay in PVR-MEF. 3D-G64S and wild-type virus were mixed in a 1:1 ratio and used to infect PVR-MEF at an MOI of 10 PFU/cell at 37 8C. At 6 h post-infection, cell-associated RNA and virus were harvested, and the virus was used to infect fresh cells at high MOI (5-30 PFU/cell). This cycle was repeated a total of three times; BstBI digestion assay products are shown for passages 1, 2, and 3 (P1, P2, P3), and the Phosphorimager-quantified percentage of the G64S product is shown. DOI: 10.1371/journal.ppat.0010011.g004 PLoS Pathogens | www.plospathogens.org October 2005 | Volume 1 | Issue 2 | e11 three different mixtures of viruses, designated by their 2C allele, depicted in Figure 5. The three mixtures were generated as follows: wild-type, containing a 1:10 mixture of wild-type and 3D-G64S virus; 2C-F28S(1m), containing a 1:10 mixture of 2C-F28S(1m) and 2C-F28S(1m)/3D-G64S virus; and 2C-F28S(3m), containing a 1:10 mixture of 2C-F28S(3m) and 2C-F28S(3m)/3D-G64S virus (

Figure 5 .
5Serial Passage Competition Assay in HeLa Cells for Mixtures of Wild-Type and 3D-G64S Viruses Containing the 2C-F28S Temperature-Sensitive Attenuating Mutation One-to-one mixtures of 3D-G64S to WT virus containing the indicated 2C-F28S mutations were used to infect HeLa cells at an MOI of 0.1 PFU/ cell at 32 8C. At 12 h post-infection, cell-associated RNA and virus were harvested, and the virus was titered to determine input for the next passage. This cycle was repeated three to five times, and representative gels of digestion products, with maps indicating the mutations at the 2C locus in the mixtures of viruses that were wild-type and 3D-G64S at the 3D locus, are shown: (A) wild-type, (B) 2C-F28S(1m), (C) 2C-F28S(3m). X denote the numbers of nucleotide substitutions at the 2C-F28S site, while O denotes the presence of the 3D-G64S mutation. DOI: 10.1371/journal.ppat.0010011.g005

Figure 6 .
6Pathogenesis of Mixtures of Wild-Type and 3D-G64S Viruses Containing the 2C-F28S Temperature-Sensitive Attenuating Mutation PVR mice were injected intramuscularly with 5 3 10 6 (A) or 2 3 10 8 (B) PFU of total virus. The viral inocula were 1/10 mixtures of the wild-type/ 3D-G64S derivatives to allow 3D-G64S viruses a chance to compete. For example, 2C-F28S(1m) was a mixture of 5 3 10 5 PFU of 2C-F28S(2C-F28S(1m) and wild-type viruses. (C) Sequence analysis of RT-PCR products from viral RNA pools (''input'') used to inoculate the mice shown in (A) and from muscle tissue after development of disease (''output''). For output samples, samples from at least three mice were sequenced, and a representative example for each is shown. DOI: 10.1371/journal.ppat.0010011.g006

Figure 7 .
7Viral Competition Assay in the Context of the 2C-F28S Temperature-Sensitive Attenuating Mutations Muscle tissue from the experiment shown in Figure 6 was processed and the BstBI digestion assay was performed. Representative gels of digestion products, with maps indicating the mutations at the 2C locus are shown: (A) wild-type, (B) 2C-F28S(1m), (C) 2C-F28S(3m). (D) Quantitation of the gels in A-C. The percent 3D-G64S is shown, and represents an average from 5-10 mice for each condition. DOI: 10.1371/journal.ppat.0010011.g007 PLoS Pathogens | www.plospathogens.org October 2005 | Volume 1 | Issue 2 | e11
PLoS Pathogens | www.plospathogens.org October 2005 | Volume 1 | Issue 2 | e11
AcknowledgmentsWe thank Jomaquai Jenkins and Elizabeth Stillman for training on animal work. We are grateful to Shane Crotty and Raul Andino for the provision of poliovirus receptor-expressing mice. This work was supported by a National Institutes of Health grant (#AI-48756), and JKP is a Rebecca Ridley Kry Fellow of the Damon Runyon Cancer Research Foundation.Competing interests. The authors have declared that no competing interests exist.Author contributions. JKP and KK conceived and designed the experiments. JKP performed the experiments. JKP and KK analyzed the data, and wrote the paper.
A virus mutations and fitness for survival. E Domingo, J J Holland, Annu Rev Microbiol. 51Domingo E, Holland JJ (1997) A virus mutations and fitness for survival. Annu Rev Microbiol 51: 151-178.

Rates of spontaneous mutation. J W Drake, B Charlesworth, D Charlesworth, J F Crow, Genetics. 148Drake JW, Charlesworth B, Charlesworth D, Crow JF (1998) Rates of spontaneous mutation. Genetics 148: 1667-1686.

The broadspectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen. S Crotty, D Maag, J J Arnold, W Zhong, J Y Lau, Nat Med. 6Crotty S, Maag D, Arnold JJ, Zhong W, Lau JY, et al. (2000) The broad- spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen. Nat Med 6: 1375-1379.

RNA virus fitness. E Domingo, L Menendez-Arias, J J Holland, Rev Med Virol. 7Domingo E, Menendez-Arias L, Holland JJ (1997) RNA virus fitness. Rev Med Virol 7: 87-96.

RNA virus error catastrophe: Direct molecular test by using ribavirin. S Crotty, C E Cameron, R Andino, Proc Natl Acad Sci U S A. 98Crotty S, Cameron CE, Andino R (2001) RNA virus error catastrophe: Direct molecular test by using ribavirin. Proc Natl Acad Sci U S A 98: 6895- 6900.

The error threshold. C K Biebricher, M Eigen, Virus Res. 107Biebricher CK, Eigen M (2005) The error threshold. Virus Res 107: 117-127.

A single mutation in poliovirus RNAdependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity. J K Pfeiffer, K Kirkegaard, Proc Natl Acad Sci U S A. 100Pfeiffer JK, Kirkegaard K (2003) A single mutation in poliovirus RNA- dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity. Proc Natl Acad Sci U S A 100: 7289-7294.

Remote site control of an active site fidelity checkpoint in a viral RNA-dependent RNA polymerase. J J Arnold, M Vignuzzi, J K Stone, R Andino, C E Cameron, J Biol Chem. 280Arnold JJ, Vignuzzi M, Stone JK, Andino R, Cameron CE (2005) Remote site control of an active site fidelity checkpoint in a viral RNA-dependent RNA polymerase. J Biol Chem 280: 25706-25716.

Poliovirus pathogenesis in a new poliovirus receptor transgenic mouse model: Age-dependent paralysis and a mucosal route of infection. S Crotty, L Hix, L J Sigal, R Andino, J Gen Virol. 83Crotty S, Hix L, Sigal LJ, Andino R (2002) Poliovirus pathogenesis in a new poliovirus receptor transgenic mouse model: Age-dependent paralysis and a mucosal route of infection. J Gen Virol 83: 1707-1720.

Transgenic mice expressing a human poliovirus receptor: A new model for poliomyelitis. R B Ren, F Costantini, E J Gorgacz, J J Lee, V R Racaniello, Cell. 63Ren RB, Costantini F, Gorgacz EJ, Lee JJ, Racaniello VR (1990) Transgenic mice expressing a human poliovirus receptor: A new model for polio- myelitis. Cell 63: 353-362.

Characterization of three different transgenic mouse lines that carry human poliovirus receptor gene-Influence of the transgene expression on pathogenesis. S Koike, C Taya, J Aoki, Y Matsuda, I Ise, Arch Virol. 139Koike S, Taya C, Aoki J, Matsuda Y, Ise I, et al. (1994) Characterization of three different transgenic mouse lines that carry human poliovirus receptor gene-Influence of the transgene expression on pathogenesis. Arch Virol 139: 351-363.

Retrograde transport of intact poliovirus through the axon via the fast transport system. S Ohka, W X Yang, E Terada, K Iwasaki, A Nomoto, Virology. 250Ohka S, Yang WX, Terada E, Iwasaki K, Nomoto A (1998) Retrograde transport of intact poliovirus through the axon via the fast transport system. Virology 250: 67-75.

Recent insights into poliovirus pathogenesis. S Ohka, A Nomoto, Trends Microbiol. 9Ohka S, Nomoto A (2001) Recent insights into poliovirus pathogenesis. Trends Microbiol 9: 501-506.

Evolution of wild-type 1 poliovirus in two healthy siblings excreting the virus over a period of 6 months. T Hovi, N Lindholm, C Savolainen, M Stenvik, C Burns, J Gen Virol. 85Hovi T, Lindholm N, Savolainen C, Stenvik M, Burns C (2004) Evolution of wild-type 1 poliovirus in two healthy siblings excreting the virus over a period of 6 months. J Gen Virol 85: 369-377.

Random selection: A model for poliovirus infection of the central nervous system. M M Georgescu, J Balanant, S Ozden, R Crainic, J Gen Virol. 78Georgescu MM, Balanant J, Ozden S, Crainic R (1997) Random selection: A model for poliovirus infection of the central nervous system. J Gen Virol 78: 1819-1828.

Trans-dominant inhibition of RNA viral replication can slow growth of drug-resistant viruses. S Crowder, K Kirkegaard, Nat Genet. 37Crowder S, Kirkegaard K (2005) Trans-dominant inhibition of RNA viral replication can slow growth of drug-resistant viruses. Nat Genet 37: 701- 709.

Genetic bottlenecks reduce population variation in an experimental RNA virus population. H Li, M J Roossinck, J Virol. 78Li H, Roossinck MJ (2004) Genetic bottlenecks reduce population variation in an experimental RNA virus population. J Virol 78: 10582-10587.

Colonization of the mouse intestine by an avirulent Salmonella typhimurium strain and its lipopolysaccharide-defective mutants. J J Nevola, B A Stocker, D C Laux, P S Cohen, Infect Immun. 50Nevola JJ, Stocker BA, Laux DC, Cohen PS (1985) Colonization of the mouse intestine by an avirulent Salmonella typhimurium strain and its lipopolysaccharide-defective mutants. Infect Immun 50: 152-159.

Role of the Salmonella abortusovis virulence plasmid in the infection of BALB/c mice. S Uzzau, P A Gulig, B Paglietti, G Leori, B A Stocker, S Rubino, FEMS Microbiol Lett. 188Uzzau S, Gulig PA, Paglietti B, Leori G, Stocker BA, Rubino S (2000) Role of the Salmonella abortusovis virulence plasmid in the infection of BALB/c mice. FEMS Microbiol Lett 188: 15-18.

Diversity at the locus associated with transcription of a variable surface antigen of Pneumocystis carinii as an index of population structure and dynamics in infected rats. S P Keely, M T Cushion, J R Stringer, Infect Immun. 71Keely SP, Cushion MT, Stringer JR (2003) Diversity at the locus associated with transcription of a variable surface antigen of Pneumocystis carinii as an index of population structure and dynamics in infected rats. Infect Immun 71: 47-60.

Succession of mutations in the Sabin strain of type 3 poliovirus replicating in the central nervous system of monkeys. Z Lu, D M Asher, I S Levenbook, K M Chumakov, Virology. 220Lu Z, Asher DM, Levenbook IS, Chumakov KM (1996) Succession of mutations in the Sabin strain of type 3 poliovirus replicating in the central nervous system of monkeys. Virology 220: 285-289.

Change in coreceptor use coreceptor use correlates with disease progression in HIV-1-infected individuals. R I Connor, K E Sheridan, D Ceradini, S Choe, N R Landau, J Exp Med. 185Connor RI, Sheridan KE, Ceradini D, Choe S, Landau NR (1997) Change in coreceptor use coreceptor use correlates with disease progression in HIV- 1-infected individuals. J Exp Med 185: 621-628.

Evidence for the HIV-1 phenotype switch as a causal factor in acquired immunodeficiency. S Glushakova, J C Grivel, W Fitzgerald, A Sylwester, J Zimmerberg, Nat Med. 4Glushakova S, Grivel JC, Fitzgerald W, Sylwester A, Zimmerberg J, et al. (1998) Evidence for the HIV-1 phenotype switch as a causal factor in acquired immunodeficiency. Nat Med 4: 346-349.

The mechanism of RNA recombination in poliovirus. K Kirkegaard, D Baltimore, Cell. 47Kirkegaard K, Baltimore D (1986) The mechanism of RNA recombination in poliovirus. Cell 47: 433-443.

Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. V R Racaniello, D Baltimore, Proc Natl Acad Sci U S A. 78Racaniello VR, Baltimore D (1981) Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proc Natl Acad Sci U S A 78: 4887-4891.


ReseARch iN Theology iN The DigiTAl Age: oppoRTuNiTies AND limiTATioNs

Research associate: Dpt. Church History and Polity
© UV/UFS <http://www.uovs.ac.za/ActaTheologica> Adriaan C. Neele
Yale University
University of the Free State

ReseARch iN Theology iN The DigiTAl Age: oppoRTuNiTies AND limiTATioNs
1015-8758D4ACCA0377BAF4337CAC9C2723D1751A10.4314/actat.v31i2.9
Digital text repositories in the field of theology and history, including the works of John calvin (1509-1564), are promising tools assisting scholars with comprehensive search capabilities, collaborative projects, annotations, and editing options.This paper discusses a case study of the opportunities and limitations of online scholarly archives of primary sources concerning the works of calvin with particular attention to research, education, and publication. 1This essay is an extended version of a paper delivered at the international congress on calvin Research, Bloemfontein, south Africa, August 22-27, 2010. 2 A digital archive is defined as a collection of digital resources, such as primary source text, focused on a specific area.primary sources are understood in this paper as a first or later letterpress or a scholarly authoritative critical edition of printed material during the era of investigation.secondary sources, then, provide interpretation and analysis of primary sources.All referenced websites have been accessed on october 10 and 11, 2011.

The phenomenal increase of digital archives in the field of humanities over the last decade has been nothing short of astounding. 2 however promising these digital repositories may seem, a critical assessment of their opportunities and limitations for research, education, and publication is indispensable.The europeana project currently includes over 15 million digital sources from over 1,000 archives in the european union (europeana 2011), the hathiTrust Digital library, which preserves and provide access to material scanned by google, the internet Archive and other partner institutions include 9 million volumes (hathiTrust 2011), and the collaborative partnership of google with the universities of michigan, harvard, and oxford has produced thus far over 15 million books online (Wikipedia 2011).Furthermore, national initiatives such as e-rara, a swiss digital library dedicated to providing free online access to rare antique swiss books and prints (e-rara 2011), and gallica, a French collaborative project (gallica 2011), contain millions of historical resources, such as books, manuscripts and maps previously only locally available.concurrent with this extraordinary increase of digital resources is the fading away of early criticism about the quality of web-based historical resources for both commercial and open-access archives -though not without facing new inquiries about the creation, dissemination and preservation of scholarly digital archives (Jones 2007  .Furthermore, many projects of digital critical editions compete constantly for sufficient grant funding to sustain an open access policy,3 or have to seek for commercial alternatives to continue their scholarly endeavors.

These trends in the digital landscape impact and increasingly will impact the way of research, education and publication of theological studies, which can be illustrated by a case-study and survey of the works of the French / swiss protestant reformer, preacher, teacher, pastor, and theologian John calvin (1509-1564).his works, in part or whole, are now available through a growing number of various portals on the worldwide web, either as open access, such as the Bibliothèque Nationale de France (gallica) (BNF), Bayerische staats Bibliothek (BSB), e-rara, internet Archive (IA), and google Books (GB), which all have been conveniently aggregated in the portal of the Post-reformation Digital Library (pRDl 2011) hosted by the h.henry meeter center for calvin studies, or subscription-based, such as the institute for Reformation Research at the Theological university of Apeldoorn (iRR), the Digital Library of Classic Protestant Texts (DlcpT), and Early English Books Online (eeBo).Although some students and scholars of Reformation studies, and calvin studies in particular, are fairly at home in the digital world, others are bemused.Therefore a brief examination of a selected number of digital primary sources of calvin, restricted to 16 th century editions -the Calvini Opera excepted, may suffice to acquaint one with the research opportunities and limitations both for open access and subscription based.

The Calvini Opera (Baum, cunitz & Reuss 1861-1900) is available at open Archive at the university of geneva (open Archive 2011) and partially at GB (google Books 2011) Access to the former initiates the downloading of the requested work -a step not required by GB, and constrained by available bandwidth.The accessed document, viewed as scanned pages of the original letterpress edition, can be searched for specific key words, whereby GB presents all search results, highlighted in context by page number, which open Archive offers one at a time.Although both venues offer download functionality (pDF format), the search function is lost with GB but remains functional in an open Archive document.Furthermore, the entry of multiple words or phrase in the search field reveals that the search engine offers proximity in a sentence or paragraph.For example, the phrase "in summa" presents search results with the words "in" and "summa" sequential or separated by words, and sentences.in other words, the accuracy of the search results requires much discernment and interpretation of the user.

The works of calvin digitally present in latin, French and english at the BNF, such as Interim adultero-germanum (calvin 1549), Defensio orthodoxae fidei de sacra Trinitate (calvin 1554), and Le Catéchisme (calvin 1552) and Sermons of upon the epistle to the Galathians (calvin 1574) are only available in black / white image format, and search by words is not possible, though download in a pDF or Jpeg format for research purposes is available to the user.Furthermore, calvin's works published in the French language are listed in the BNF but originates from e-rara calvin 1548) and the BSB (calvin 1538). 4it presents the page image in color, and is non-searchable but downloadable, whereby the BSB contain works translated into german, and both contain various commentaries, sermons, treatises and other works.Finally, the IA offers assorted calvin works not only in various online formats, such as viewer, pDF -color and black / white, e-pub., Kindle, Daisy, DjVu and full text, whereby the read-online and both pDF versions are searchable -a functionality that remains after the pDF downloading.These works then, available through open access, offer an entry globally to the rare editions of calvin's work, which were previously only available locally. in some cases the work is presented with key word search functionality, and in other cases with high-quality digital scans in color -a laudable attempt to embody the original book enhancing the use of these digital primary sources.Furthermore, portals such as the PRDL, an aggregated website, greatly improve the accessibility, and foster a guided use of the various digital resources of calvin's work such as commentaries, sermons, doctrine, polemic, catechetical / liturgical, and correspondence (pRDl 2011).

suBscRipTioN-BAseD: DlcpT, eeBo AND iRR

The DLCPT offers more than sixty selected works of calvin, which are fully and advanced searchable, such as single term and phrase search, proximity searching in the same sentence or in the same paragraph on selected parts and the entire work or collective calvin corpus; each work containing page images of the original letterpress edition connected to the searchable digital edition.search results are presented in context or line by line, and in page order, and can be viewed by individual page, paragraph or section.The accuracy of the search result is highly precise, and offers the user the option to view the corresponding page image of the printed work in a separate window.page images of printed works, though with various image quality and limited search capabilities is the core of the more than fifty english works of calvin offered by EEBO.The Calvini Opera (CO) developed by iRR as fully searchable pDF's on compact disc formed the basis of a recent and promising initiative -the combination of a fully and advanced searchable CO integrated with corresponding page image, like the DLCPT, and whereby specific works can be linked to digital editions that are available as open access documents.The user is then able to view the search result of, for example, the Interim Adultero-Germanum, as digital text, text image of the CO and text image of the 1549 edition available through BNF (calvin 1549).Furthermore, iRR envisions that the CO is cross-searchable with other series of the Corpus Reformatorum, and other works of the sixteenth-century Reformation.

These works of calvin, then, differentiate themselves primarily in their advance search capabilities and the level of precision of search results from the current open-access publications, as well as their ability to view the digitally formatted text with the corresponding page image. in this way, EEBO offers download functionality for licensed users, which option is currently not available by DLCPT and iRR.This succinct survey shows the various and diverse digital primary sources of the works of this 16 th -century protestant Reformer.Although the majority of the commercially available digital works are still available only through sometimes cost-prohibitive user licensing, a more recent trend shows that open-access initiatives such as GB, IA, and e-rara make identical editions available.For example, the 1540 Commentarri in Epistolam Pauli as Romanos, previously available digitally through the DlcpT, is now freely accessible through IA (calvin 1540).other publications, such as the Defensio othodoxa fidei de sacra Trinitate, are now available through the DLCPT, BNF and GB. 5 here, two observations are in order: first the search limitation of the open access archives remains a factor, but this might be temporary given the strong development efforts of advanced optical character recognition technology, including accurate identification of 16 th century letter types.however, if this trend continues, which is not unlikely, only the differentiating search functionality, quality of images, and user options could justify the continuation of restricted access.Furthermore, this trend shows also the complexity of the digital world, which may offer various outcomes for the deployment of these digital primary sources in research, education, and publication.concerning research, the current, and ever-increasing, number of available web-based primary sources present scholars and students of calvin studies with advantages, such as local and round-the-clock availability of primary sources, building a personal library of digital sources for research purpose, and speedy locating of certain text(s) or phrases, previously part of a painstaking and time-consuming labor.however, these quickly generated search results must not only be judged on the accuracy of the primary source text but also on their lack in general of the surrounding text and accompanying argument of the author that is present in reading a tome physically in hands.Furthermore, the presentation of multiple search results of a certain word or phrase may assist the user in identifying possible connections of the author's flow of argument, yet one should exercise caution in constructing any initial conclusions from such search results (hatch 2007:150-160; maynard & o'Brien 2010:400).For example, a key word search in calvin's Psychopannychia (1534) on "anima" provides sixty-five occurrences in paragraph context, and reveals calvin's use of the word in various ways.The search, however, neither takes into account the various declensions of the word, nor do the results give an immediate sense of the relationship, if any, to the main argument of the treatise.in short, the gain of access of the digital primary source may be tempered by the quality of the retrieved information, whereby the task of critical understanding and interpretation of the text remains with the reader.in regard to education, those that teach calvin studies should consider incorporating these digital primary sources in course curricula.Although the students, the next generation of calvin scholars, are usually more acquainted with the digital world than the one currently teaching the subject matter, the exposure to primary source material offers new teaching opportunities.This is especially true for those countries that previously have been deprived of primary sources, and the ability to identify, contextualize, explore, analyze and evaluate the source (Walbert 2004; Anderson 2009:35-38; Johnston 2009:50-52; Ridgway 2009:60-61).The latter may be enhanced by comparing (digitally) such source with later editions, critical editions, and secondary sources.For example, calvin's Acta Synodi Tridentinae is present at GB as the critical edition, the CO (7:365-506), whereby the text is accompanied with explanatory or interpretive notes, but also available as the 1547 edition at the BSB and IA, without editorial apparatus (calvin 1547).such may assist students develop a more precise skill of a discerned reading of the primary text and editorial commentary, and enhance the quality of learning -though such learning does not ignore the pedagogical and didactical expediency of nonlinear reading of the codex book that so amazed st.Augustine (grossman 2011:13).Furthermore, the increased use of e-learning applications offers ample opportunity to integrate the digital primary sources of calvin's works into the study curriculum, which in that way also may contribute to a more eco-friendly learning environment by reduced paper use.

Finally, the use of digital primary sources will test new ways of citations, annotation, and open venues for publication in various forms such as e-journals and e-books, but also will present challenges of copyright and licensing guided or not by fair-use provisions.Despite these challenges, the institut für schweizerische Reformationsgeschichte, in conjunction with the Zwingliverein, recently launched a comprehensive, yet user-friendly academic and peer-reviewed e-journal (Zwingliana).Authors have the option to submit articles, and users can register to have free access to electronic published issues with a three year delay and will find more than 2,000 full text searchable articles in over 200 different issues of the journal since its founding in 1897.This form of publication, dedicated to calvin studies, may foster an additional way of scholarly communication. 6 coNclusioN in conclusion, the current developments in the digital landscape, including the field of theology, and in particular those related to the studies of calvin's work by a continuing and increasing number of available primary sources, as well as a rising number of possibilities of making use of these sources, offer at the same time various opportunities and limitations, as the boundaries of scholarship, open access and commercial interest will continue to be tested, appropriated and defined.The rising number of early editions of the geneva reformer available through open access shows concurrently a duplication of digitalization effort.Although the cost of digitalization may continue to decrease over time, a coordinated and cooperative initiative may lead to making more works available, and optimizing hosting space and preservation.7 Furthermore, researchers should continue consulting commercial digital archives of calvin resources, leveraging publications and editions that are not available through open access and making use of the advanced search capabilities such archives offer. in addition, the combination of different digital primary sources other than calvin's text, such as, for example, digital cartography, historical records of the city of geneva, and sixteenth-century publishers and printing houses may advance cross-disciplinary research, especially when crosscollection searches can retrieve collective results from such digital archives.Digital scholarly archive projects, such as First source at the Jonathan edwards center at yale university, promise advanced interplays between the Calvini and Melanchtoni Opera, the edwards corpus, and Westminster Assembly records, thereby opening new ways of comparative research (First source 2011).

The field of theology should make discriminated use of the new possibilities of digital research, education and publication to raise academic standards by internationally advancing thoughtful theological reflections through digital media.
 open access (publishing) is defined as access to material (mainly scholarly publications) via the internet in such a way that the material is free for all to read, and to use (or re-use) to various extents.
This site offers various forms of locating a document such as browsing-index, name, period, and key word search, as well as translated works.
An overview of all open access and commercial available works of calvin can be obtained from the author at email@example.com.
Open Journal Systems is an open source journal application and a viable candidate to implement an e-journal for calvin studies.

instituut voor Reformatie onderzoek, h. henry meeter center for calvin studies, and institut für schweizerische Reformationsgeschichte may consider to lead such initiative. J Allinson, S Francois, MultiMedia & Internet@Schools. cAlvin, J. 15381662008. 2009. de%2F~db%2Fmets%2Fbsb00027625_metssWoRD: simple Web-service offering Repository Deposit Ariadne

Les Actes du Concile de Trente avec le remède . . . geneva: Jean girard.

sur l'Epistre aux Ebrieux. geneva: Jean girard. Commentaire,

Interim adultero-Germanum, cui adiecta est vera Christianae pacificationis, et ecclesiae reformandae ratio. geneva: Jean girard.

Institutio totius christianae religioni. . . geneva: Jean girard.

De Scandalis, geneva: Jean crespin.

Catéchisme : c'est à dire, le formulaire d'instruire les enfants en la chrestienté, fait en la manière de dialogue, où le ministre interrogue et l'enfant respond. geneve: iehan crespin. 1554. Defensio orthodoxae fidei de sacra Trinitate, contra prodigiosos errores Michaelis Serveti. geneva: oliua Roberti stephani

Von der Papisten Heiligthumb. pforzheim: g Raben.

Summarischer Begriff der lehr von den heiligen Sacramenten. heidelberg: J. maier.

Calvine upon the epistle of saincte Paule to the Galathians. london: lucas harison. M Sermons, John, 1574

Iohannis Calvini vber das buch Job. herborn, c. Raben. H Predigten,

oclc offers long-Term Digital Archive storage. Sermons De, M Jean Calvin Sur Le V ; Feijen, M Horstmann, W , 10.3931/e-rara-1057AAiAAJ&printsec=frontcover#v=onepage&q&f=false> digitAl librAry oF clAssic ProtestAnt texts 2011. E Baum, E Cunitz, Reuss, Netherlands, The hague2011. 2007. 2008133Isis. hAthi trust digitAl librAry 2011. hathi Trust Digital library. r. & PoWell, A. 2006. Digital Repositories Roadmap: looking forward, JISC. [online

A historical partnership: Teaching with primary sources. M P Johnston, School Library Monthly. 2642009

Institutional Repositories: Content and Culture in an Open Access Environment. oxford: chandos publishing. C Jones, Science and Technology Facilities Council e-Science. 2007. 2008. Nov.Report of the subject and institutional Repositories interactions study

scholarly output: print and digital in teaching and research. S Maynard, A & O'brien, Journal of Documentation. 6634002010

Teaching with primary sources: professional Development from the library of congress at your Fingertips. oPen JournAl systeMs 2011. public Knowledge project. simon Fraser university Vancouver. 2009. 200638Reading primary sources: An introduction for students

lisT oF ABReViATioNs useD BNF -Bibliothèque Nationale de France BsB -Bayerisches staats Bibliothek DlcpT -Digital library of classic protestant Texts eeBo -early english Books online gBgoogle Books iAinternet Archive. WikiPediA 2011. google Books. Reformation studies institute, university of Zurich