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1 Center for Molecular Chaperones/Radiobiology and Cancer Virology, Medical College of Georgia, Augusta, GA 30912, USA
2 Molecular Integrative Neuroscience Department (MIND), The Scripps Research Institute, La Jolla, CA 92037, USA
Correspondence
Demetrius Moskophidis
dmoskophidis{at}mcg.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work. 
Present address: Office of Cellular, Tissue and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike HFM 730, Rockville, MD 20852-1448, USA.
Supplementary material is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). They are rodent-associated viruses, with the single exception of Tacaribe virus, which infect bats and their geographical distribution is determined by the range of the reservoir species. The worldwide distribution of LCMV reflects its ubiquitous natural reservoir, Mus musculus. In contrast, other arenaviruses are endemic to specific geographical regions including SubSahara West Africa (Lassa virus) and South America (Junin, Machupo, Guanarito and Sabia viruses). Human infections occur from inhalation of aerosolized virus or by direct contact with contaminated material (Damonte & Coto, 2002; Jay et al., 2005; Peters, 2002). Despite the intense study of arenavirus biology since the isolation of the first family member, LCMV, in the 1930s, there is not much understanding of the molecular determinants of arenavirus-induced disease in humans and animals.
Arenaviruses are enveloped viruses with a bi-segmented negative-strand RNA genome consisting of a small (S) and a large (L) segment with approximate sizes of 3.4 and 7.2 kb, respectively (Meyer et al., 2002; Romanowski & Bishop, 1985). Each RNA segment has an ambisense coding strategy, encoding two proteins in opposite orientation separated by an intergenic region (IGR). The S RNA directs synthesis of the nucleoprotein (NP) (ca. 63 kDa) and two mature virion glycoproteins, GP1 (40–46 kDa) and GP2 (35 kDa), derived from post-translational cleavage of a precursor polypeptide, GP-C (75 kDa). GP1 and GP2 make up the spikes on the virion envelope. GP1 mediates the virus interaction with its host cell receptor, identified as 
-dystroglycan for Lassa and Old World viruses, or transferrin receptor 1 for the New World arenaviruses (Cao et al., 1998; Kunz et al., 2002; Radoshitzky et al., 2007). The L RNA segment encodes the viral polymerase (L; ca. 200 kDa) and a small polypeptide Z (ca. 11 kDa) containing a zinc-binding RING finger motif. Evidence indicates that Z is the arenavirus counterpart of the matrix (M) protein found in many negative-RNA viruses (Perez et al., 2003).
LCMV provides investigators with a superb model for the investigation of viral immunology and pathogenesis (Ahmed et al., 1996; Hotchin, 1971; Lehmann-Grube, 1972; Oldstone, 2006; Traub, 1936; Zinkernagel et al., 1993). In particular, LCMV infection in the mouse has been used to study the dynamics of virus–host interactions in the context of viral persistence, uncovering two extreme scenarios regarding the interaction between the virus and the host immune system. The first is that a sustained CD8+ T cell-mediated response results in virus clearance within 2 weeks after infection. The second situation involves a transient CD8+ T-cell response, in which antigen-specific CD8+ T cells are induced and proliferate, initially exhibiting antiviral function but progressively losing this ability (clonal exhaustion), which leads to viral persistence (Moskophidis et al., 1993). Such functionally deficient T cells persist in the host for long periods but may eventually be eliminated (Zajac et al., 1998; Zhou et al., 2004). Both outcomes (viral clearance or persistence) are of limited pathological consequence for the host and are determined by the strength and magnitude of the virus-specific immune response and the rate of virus replication (Leist et al., 1988; Moskophidis et al., 1995; Thomsen et al., 1996). Thus, fast-growing LCMV isolates, such as Docile (Doc) strain, readily induce persistent infection, whereas the slower replicating Aggressive (Agg) isolate does not (Ahmed et al., 1984; Moskophidis et al., 1995; Pfau et al., 1982). Notably, both Doc and Agg isolates were derived from the same parental UBC strain of LCMV, suggesting high genomic similarity between these two strains and hence providing a valuable model for studying viral determinants of persistent infections. In this study, we provide detailed genetic and biological characterization of Doc and Agg strains with respect to their differential ability to cause persistent infections to elucidate the basis of persistent infections and associated diseases caused by LCMV in its natural host.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Viruses.
LCMV-Doc and -Agg (isolated from an LCMV-UBC-carrier mouse) were obtained from Dr C. J. Pfau (Department of Biology at Rensselaer Polytechnic Institute, Troy, NY, USA) (Pfau et al., 1982). LCMV-Armstrong was obtained from Dr Rafi Ahmed (Emory University Vaccine Center, Atlanta, GA, USA). LCMV titres were determined with an immunological focus assay (Battegay et al., 1991).
Assessment of virus-specific T cell-mediated immune response.
Gamma interferon (IFN-
) intracellular staining and major histocompatibility complex class I tetramer staining were performed as described previously (Ou et al., 2001) (details in Supplementary Material available in JGV Online).
Delayed type hypersensitivity (DTH) reaction.
Virus-specific DTH reaction was determined as local swelling after subcutaneous inoculation of virus in the footpad as described previously (Moskophidis & Lehmann-Grube, 1989).
Details of the following Methods are available as Supplementary Material in JGV Online. Sequence analysis of LCMV Docile and Aggressive, Analyses of the S and L segment genome of Doc or Agg 5' terminal sequences, Population analysis of the 5' and 3' genomic termini of S and L RNA fragments of LCMV strains Docile and Aggressive, Construction of plasmids and transfection, Verification of viruses recovered from cDNA and Isolation of biological reassortant viruses containing the S or L RNA segment of Doc or Agg.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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and Supplementary Figs S1, S2, S3 and S4) [GenBank accession no. EU480450
[GenBank]
(Agg S segment), EU480451
[GenBank]
(Agg L segment), EU480452
[GenBank]
(Doc S segment), EU480453
[GenBank]
(Doc L segment)]. We identified 14 sequence substitutions from a total of 3377 nt in the S RNA that resulted in nine predicted amino acid differences, four in GP and five in NP coding regions. In the L RNA sequence, we found 42 nt substitutions from a total of 7227 nt, that resulted in 13 predicted amino acid differences within the L protein between Doc and Agg, whereas no coding substitutions were found within the Z protein. Within the non-coding (5', 3' and IGR) regions of the S RNA of Doc and Agg we observed only 1 nt difference in the IGR, which was outside of the predicted RNA stem–loop structure. The alignment of the non-coding regions of Doc and Agg L RNA segments revealed identical 5' and 3' ends, but both viruses differed at 6 nt positions within the IGR, including three in the predicted secondary RNA structure.
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). Our results revealed that the viral RNA population was composed of a mixture of RNAs with full-length termini and RNAs with deletions, mostly of 1–4 nt within the conserved 5' or 3' end sequences as reported for LCMV Arm (Meyer & Southern, 1997). In particular, we observed that a subset of truncated L RNAs exhibited larger (14–34 bases) deletions at their 5' and 3' termini. Analysis of the S and L RNA populations carried out by a 3'–5' RNA junction-length spectra-analysis, a technique broadly similar to that used to analyse the CDR3 distribution of T-cell receptors in a T-cell population (Pannetier et al., 1992), revealed diversity in the viral RNA population (S or L RNA) for both Doc and Agg (data not shown). Since the sizes of deletions in the S and L RNA termini were similar for both virus strains, it is unlikely that the presence of such deletion variants in the viral pool contributed to the Doc strain's ability to cause persistence.
It has been demonstrated that arenavirus S and L segments carry out a non-templated G residue at their 5' ends (Flatz et al., 2006; Garcin & Kolakofsky, 1990; Meyer & Southern, 1997). However, it was impossible, using the RNA ligation method, to clearly identify whether the 5' ends of the viral segments of Agg and Doc carry out a non-templated G. Therefore, we analysed the viral S and L segment 5' ends using a 5' RACE protocol (for details see Supplementary Material available in JGV Online). This approach clearly demonstrated a G residue at position –1 on both the S and L segment genomes of LCMV-Doc and -Agg (Fig. 1b
). In addition, we observed that both the S and L RNA 5' ends contained variably an additional T residue at position –2. Likewise, sequencing of individual cDNA clones confirmed this result, but revealed that some clones contained one or two additional nucleotides at positions –3 and –4. Based on our data and that in the literature the 3' ends of viral genome segments terminate on a G residue. Thus, we suggest that in analogy to other arenaviruses (Garcin & Kolakofsky, 1990), the genome of Doc and Agg share common terminal nucleotide sequence and that intramolecular annealing of their genome into a panhandle structure does not form a flush end.
Recovery of infectious LCMV from cDNA by RNA polymerase I/II-based reverse genetics
To further explore the molecular basis underlying the ability of the Doc strain to cause a persistent infection, we used a pol I/II-based reverse-genetics system to generate isogenic infectious recombinant LCMV for the Agg, Doc or Arm strains (Fig. 2a and b). Co-transfection of BHK-21 cells with four plasmids (pol I-SAgg, pol I-LAgg, pC-NPAgg and pC-LAgg) resulted in recovery of infectious virus from the culture supernatant within 3–4 days. Likewise, this pol I/II-driven system was efficient in recovery of Doc and Arm from cloned cDNA. We also rescued Agg and Doc recombinant viruses by co-expressing the trans-acting factors L and NP from Arm together with the corresponding pol I S and L plasmids. This finding was consistent with the observed high frequency of reassortant viruses between genetically closely related arenaviruses. Using the same approach, we generated reassortant viruses containing the same L segment of Agg but with the S segment of Doc or Arm (list of rescued virus strains in Fig. 2c). The genetic identity of the rescued Agg (rAgg), Doc (rDoc), Arm (rArm) and reassortant viruses was confirmed by sequencing RT-PCR-amplified DNA fragments from selected regions of the genome. In addition, we also used restriction enzyme digestion of RT-PCR-generated DNA fragments spanning selected regions of the viral S or L RNA segments (Fig. 2d). Cloned viruses rescued from cDNA formed plaques on Vero cells of comparable size to those formed by the authentic wild-type Agg (Agg-wt), Doc (Doc-wt) or Arm (Arm-wt) viruses. Both Arm-wt and rArm had similar growth properties in cultured cells, whereas rDoc and rAgg grew at slightly higher rates compared with Agg-wt or Doc-wt strains (Fig. 2e). This was attributed to the identical genetic makeup of rAgg and rDoc virus (potentially free of defective interfering particles).
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). As anticipated, viral titres peaked between days 3 and 6, followed by a rapid decline below detectable levels by day 15 post-infection (p.i.) in all tissues examined (shown for spleen but also studied in liver and kidney). However, we noted that clearance of rAgg occurred with slightly faster kinetics compared with Agg-wt, whilst significant differences were not observed in the kinetics of viral replication and clearance between rDoc and Doc-wt. In addition, whilst both cDNA-derived and wild-type virus strains induced efficient virus-specific CD8+ T-cell responses, mice infected with rAgg exhibited higher levels (3–5 fold) of virus-specific CD8+ T-cell response at days 6 and 9 p.i. compared with Agg-wt-infected mice, which may explain the faster kinetics of viral clearance. No significant difference in the magnitude of antiviral CD8+ T-cell responses was observed between Doc-wt- and rDoc-infected mice.
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-production) deficit (Fig. 4 and data not shown). In contrast, this response was suppressed in mice persistently infected with either rDoc or Doc-wt, and in both cases infected mice exhibited similar kinetics of CD8+ T-cell exhaustion.
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; Moskophidis & Lehmann-Grube, 1989). rAgg, rDoc and rArm viruses induced footpad swelling reactions of similar duration and magnitude, comparable to those produced by animals inoculated with Doc-wt, Agg-wt or Arm-wt (Fig. 5). LCMV persistence is known to suppress T cell-induced local inflammation (DTH reaction) (Moskophidis et al., 1995). To test whether the cDNA-derived viruses shared the ability of their wild-type strains to cause acquired T-cell suppression, we infected mice locally (i.f.) with 104 p.f.u. of rDoc or rAgg virus followed by infection intravenously (i.v.) with increasing amounts of the same virus. rDoc-infected mice mounted a footpad swelling response that was substantially suppressed at a dose of 102 p.f.u. and was below detection levels at a dose of 104 p.f.u. (i.v.). In contrast, inoculation of mice with rAgg virus did not significantly affect footpad swelling at a dose of 102 p.f.u. and only partially at a dose of 104 p.f.u., but the response was suppressed at high doses (106 p.f.u., i.v.) (Fig. 5b and c). Together, our results strongly indicate that rDoc and rAgg recreated accurately the phenotypic properties of the corresponding bona fide Doc-wt and Agg-wt viruses.
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secretion. rArm- or Arm-wt-infected mice mounted virtually unimpaired CD8+ T-cell responses that resulted in rapid viral clearance by day 9 p.i. from different tissues (spleen, liver and kidney). Notably, rSDocLAgg-infected mice mounted an unimpaired CD8+ T-cell response that suppressed virus replication below detectable levels at days 15–20 p.i. This result revealed that the S segment of Doc does not contain all the genetic determinants responsible to cause virus-specific CD8+ T-cell exhaustion and establish persistence. The fact, however, that the reassortant rSArmLAgg virus exhibited a significantly attenuated phenotype compared with rAgg suggests that the S RNA structure may also influence viral properties and thus the viral persistent phenotype, a view strongly supported by the LCMV literature (Matloubian et al., 1993; Mueller et al., 2007; Salvato et al., 1991). Thus, a high dose of rArm/LAgg virus was efficiently controlled by day 9 p.i. by a robust virus-specific CD8+ T-cell response that developed with kinetics comparable to that produced by infection with Arm.
To obtain a more comprehensive analysis of the in vivo phenotype of the cDNA-derived viruses, we investigated the effects of viral load on the overall outcome of infection and parameters of the virus-specific CD8+ T-cell response on day 30 p.i., at a time when the outcome of infection was determined. Our data (Fig. 6) further confirmed that the phenotypic differences between Doc and Agg viruses hold true for rDoc or rAgg. Thus, rDoc exhibited a greater ability to induce functional exhaustion of virus-specific CD8+ T-cell populations (see arrows), which could be observed at a moderate virus dose of 104 p.f.u. In contrast, the suppressive capacity of rAgg was limited, and even a high dose of 2x106 p.f.u. was efficiently cleared, despite an observed partial suppression of virus-specific CD8+ T-cell response. Notably, the presence of the S RNA segment of Doc in the rSDocLAgg reassortant did not convert the non-persistent Agg to the persistent Doc. However, we noted that the rSDocLAgg virus exhibited a moderate increased ability to suppress the NP396-404-specific CD8+ T-cell function. This finding suggests that the S RNA alone may exert an effect on the persistent phenotype. Consistent with the high dose infection results (Supplementary Fig. S5), the presence of the Arm S RNA in rSArmLAgg virus abolished the ability of rAgg to induce a partial suppression of the virus-specific CD8+ T-cell response even when injected at high dose, and it substantially attenuated viral spread. Taken together, the persistent phenotype is at least determined by the nature of the L RNA segment. However, a second cloned reassortant virus (rSAggLDoc) will demonstrate the importance of the S RNA segment in viral persistence. However, the recovery of this reassortant virus strain via reverse genetics has proven to be a challenging task for unknown reasons.
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) and performed analyses evaluating their ability to subvert an antiviral CD8+ T-cell response and persist. Since the amino acid substitution GP280N
S in Doc, not present in the Agg strain, abolishes CD8+ T-cell recognition of the GP276-286 peptide and may contribute to the persistent phenotype of this virus, we isolated two sets of reassortant viruses (Fig. 7b): virus isolates that contain the S RNA of Agg-variant (GP280NY)(Agg280Y) and Doc L RNA segments and the reverse, or viruses with the S RNA of Agg with Doc revertant (GP280SN) (Doc280N) L RNA segments. Note that the Doc280N has been isolated from infected mice and exhibits sequence similarity with wild-type or rDoc in the coding region of GP and NP proteins except for the coding change (GP280SN) that restored the GP276-286 epitope. In addition, the Agg280Y variant isolated using standard CTL selection procedures (Aebischer et al., 1991) lacks CTL recognition on the GP276-286 epitope. The fact that both virus strain combinations (Agg280Y vs Doc or Doc280N vs Agg) exhibit identical recognition patterns in this major CD8+ T-cell epitope allows viral properties associated with the persistent phenotype to be more accurately mapped to viral RNA segments. The growth rates of reassortant viruses in BHK-21 cell cultures were found to be comparable to the parental viruses (Fig. 7c).
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and Supplementary Fig. S6, we investigated the effects of viral load on the overall outcome of infection and parameters of the virus-specific CD8+ T-cell response. Collectively, our data further confirmed and extended our results with rSDocLAgg virus that the presence of the S or L RNA segment of Doc in the SDocLAgg280Y or SAgg280YLDoc reassortants did not convert the non-persistent Agg to the persistent Doc. Similar analyses performed with the Doc280N, Agg and SDoc280NLAgg viruses (Supplementary Fig. S6) confirmed the result with rSDocLAgg. However, we noted that restoration of the GP276-286 epitope in Doc280N measurably diminished its dose-dependent ability to suppress the CD8+ T-cell response causing persistent infections. This finding suggests that the substitution 280NS in the viral GP contributes to the persistent phenotype of Doc. Collectively, the data suggest that the nature of both S and L RNA segments contribute to the Doc persistent phenotype.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Evidence indicates that amino acid substitutions in the viral S and L RNA segments can affect the ability of LCMV to persist by different mechanisms including: (i) reduced virus recognition by T cells (Lewicki et al., 1995; Moskophidis & Zinkernagel, 1995; Pircher et al., 1990); (ii) changes in virus–receptor interactions and cell tropism (Sevilla et al., 2004); and (iii) changes in virus replication and overall viral load (Sevilla & de la Torre, 2006). The limited genetic diversity we found between the Doc and Agg strains facilitates studies aimed at assessing the contribution of specific amino acid residues to the persistent phenotype associated with the Doc strain. Of particular interest is a recent report that provided a comprehensive screen of the entire LCMV Arm proteome for CD8+ T-cell epitopes (Kotturi et al., 2007). Among a total of 28 CD8+ T-cell epitopes identified, nine are located in the GP, four in the NP and 15 in the L protein. Whether all of these T-cell epitopes are recognized in Doc- and Agg-infected cells remains to be determined. Comparison of the Doc versus Agg proteomes revealed a single amino acid difference in three of 28 epitopes (all within the GP), a result that facilitates correlation of host immune recognition patterns with the virus' ability to persist. For example, the substitution 280NS in the GP2276-286 T-cell epitope of Doc abolishes CD8+ T-cell recognition, and our data indicate that this can influence the persistent viral phenotype. To what extent additional coding changes found in the GP of Doc may influence the viral phenotype remains to be determined. The coding change 494VI found adjacent to the WKRR sequence motif at the C terminus of GP2 may influence the structure and function of the GP ectodomain. This alteration could affect the proteolytic processing of the viral GP, which is critical for virus production, or modulate interactions of GP with cellular factors that play roles in oligomerization of the GP (Kunz et al., 2003). The contribution to the persistent Doc phenotype of the five residual differences between the NP of Doc and Agg strains remain to be determined. NP plays a critical role in formation of RNPs and control of the replication and transcription of the virus genome (Lee et al., 2000). It is therefore plausible that these changes in the NP of Doc may contribute to increase virus growth in mouse tissues, which may represent a parameter for viral persistence. The possibility that NP mutations may interfere with the innate response (e.g. type I IFN) (Martinez-Sobrido et al., 2006; Moskophidis et al., 1994) deserves particular consideration. Thus, an amenable approach to address the biological implication of the IFN-inhibiting activity of NP of Doc and other LCMV strains is to generate recombinant LCMV viruses in which the NP genome is modified (mutated or replaced with NP sequence of other LCMV strains) and examine their biological properties in the context of both acute and persistent LCMV infection.
The in vivo phenotypes of the rSDocLAgg or biological reassortants support a critical contribution of the L segment to the persistent phenotype. Mutations in the L protein may modulate polymerase activity associated with viral genome replication and transcription. We cannot yet propose a specific role for any of the several amino acid differences scattered throughout the entire sequence of the L gene of Doc versus Agg. Notably, we did not observe sequence differences between Doc and Agg for any of the 19 CD8+ T-cell epitopes described in the L protein of Arm. Finally, the IGR is a bona fide transcription termination signal and appears to be involved in viral packaging (Pinschewer et al., 2005). Therefore, it is possible that the 5 nt differences found between the IGRs of Doc and Agg strains might contribute to the phenotypic differences between these two viruses.
The reverse-genetics approach presented here and used by others to rescue Arm and Arm Cl-13 LCMV strains from cloned DNA (Flatz et al., 2006; Sanchez & de la Torre, 2006) can facilitate the investigation of complex issues about virus structure, virus–cell interactions and viral pathogenesis, and should facilitate the development of attenuated vaccines to combat arenaviral infections. Notably, the data presented here add to the list of published studies with reassortants of different arenavirus strains, which have evaluated viral determinants associated with the virulence and pathogenicity of particular virus strains (Bergthaler et al., 2007; Lukashevich, 1992; Lukashevich et al., 2005; Matloubian et al., 1993; Oldstone et al., 1990; Riviere et al., 1985; Zhang et al., 2001). Generation of recombinant Doc viruses containing coding mutations corresponding to those of the Agg virus strain and their phenotypic characterization in vivo will provide new insights into the mechanisms underlying the ability of the Doc strain to cause persistent infection by driving virus-specific T cells into distinct programmes of clonal exhaustion.
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ACKNOWLEDGEMENTS |
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Received 20 September 2007;
accepted 14 February 2008.
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, 
, 
, 
and 
), and wild-type (authentic) viruses (Agg-wt, Doc-wt or Arm-wt) indicated by open symbols (
, 
and 
). Kinetics of virus growth were determined on BHK-21 cells infected at an m.o.i. of 0.1 or 0.01. Data shown are mean±SEM log10 p.f.u. ml–1 and are representative of three experiments.
