Characterization of an infectious cDNA copy of the genome of a naturally occurring, avirulent coxsackievirus B3 clinical isolate

C.-K. Lee1,{dagger},{ddagger}, K. Kono1,{dagger}, E. Haas2, K.-S. Kim1, K. M. Drescher3, N. M. Chapman1 and S. Tracy1

1 Enterovirus Research Laboratory, Department of Pathology and Microbiology, University of Nebraska Medical Center, 986495 Nebraska Medical Center, Omaha, NE 68198, USA
2 Department of Pathology and Microbiology, University of Nebraska Medical Center, 986495 Nebraska Medical Center, Omaha, NE 68198, USA
3 Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178, USA

Correspondence
S. Tracy
stracy{at}unmc.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Group B coxsackieviruses (CVB) cause numerous diseases, including myocarditis, pancreatitis, aseptic meningitis and possibly type 1 diabetes. To date, infectious cDNA copies of CVB type 3 (CVB3) genomes have all been derived from pathogenic virus strains. An infectious cDNA copy of the well-characterized, non-pathogenic CVB3 strain GA genome was cloned in order to facilitate mapping of the CVB genes that influence expression of a virulence phenotype. Comparison of the sequence of the parental CVB3/GA population, derived by direct RT-PCR-mediated sequence analysis, to that of the infectious CVB3/GA progeny genome demonstrated that an authentic copy was cloned; numerous differences were observed in coding and non-coding sequences relative to other CVB3 strains. Progeny CVB3/GA replicated similarly to the parental strain in three different cell cultures and was avirulent when inoculated into mice, causing neither pancreatitis nor myocarditis. Inoculation of mice with CVB3/GA protected mice completely against myocarditis and pancreatitis induced by cardiovirulent CVB3 challenge. The secondary structure predicted for the CVB3/GA domain II, a region within the 5' non-translated region that is implicated as a key site affecting the expression of a cardiovirulent phenotype, differs from those predicted for cardiovirulent and pancreovirulent CVB3 strains. This is the first report characterizing a cloned CVB3 genome from an avirulent strain.

{dagger}These authors contributed equally to this work.

The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY673831.

{ddagger}Present address: Department of Laboratory Medicine, Korea University Guro Hospital, Guro-gu Guro-dong 80, 152-050 Seoul, Republic of Korea.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Group B coxsackieviruses (CVB1–6) are enteroviruses in the species Human enterovirus B (HEV-B), family Picornaviridae (Pallansch & Roos, 2001). The CVB genome comprises a positive-stranded RNA that is 7400 nt long; the single ORF encodes a 2200 aa polyprotein that is processed during translation by two viral proteases. The ORF is flanked by 5' and 3' non-translated regions (NTRs), which encode no proteins, but are highly structured RNA sequences that are important for virus replication (Jackson & Kaminski, 1995; Liu et al., 1999). CVB cause diverse human diseases, including myocarditis, pancreatitis, aseptic meningitis and possibly type 1 diabetes (Bowles et al., 1986; Disney et al., 1953; Hyoty et al., 1998; Imrie et al., 1977; Kaplan et al., 1983; Kennedy et al., 1986; Longson et al., 1969; Martino et al., 1995; Melnick et al., 1949; Melnick, 1996; Mertens et al., 1983; Sigurdsson & Bækkeskov, 1990; Smith et al., 1998). No vaccines exist for enteroviruses except for the poliovirus (PV) vaccines (Minor, 2003). However, different attenuating approaches have been studied for the CVB (Chapman et al., 2000b; Gauntt et al., 1983; Tu et al., 1995; Willian et al., 2000), which demonstrate the potential of CVB-based vaccines and/or expression vectors (Chapman et al., 2000a; Henke et al., 2001; Höfling et al., 2000).

CVB3 strains can be myocarditic (inducing both myocarditis and pancreatitis in mice) or pancreovirulent (inducing only pancreatitis) (Tracy et al., 2000). Assays of diverse CVB3 strains in mice, isolated between the early 1950s and the late 1990s, suggest that avirulent strains are rare (Tracy et al., 2000; S. Tracy, unpublished data). It is unknown whether this is due to a sampling bias, as most CVB strains are isolated from suspected cases of viral disease and thus would be expected to express a virulent phenotype, or to other reasons. The viral genes that determine naturally occurring CVB virulence phenotypes remain unclear. By using swine vesicular disease virus (SVDV), a porcine enterovirus that is postulated to have originated from a CVB5 infection of pigs (Brown et al., 1973; Knowles & McCauley, 1997), Kanno and colleagues mapped SVDV virulence to capsid protein VP1 and protease 2A, identifying two sites (VP1 aa 132 and 2Apro aa 20; Kanno et al., 1999). Both sites affected virulence; mutation of individual sites did not have the influence of both sites mutated at the same time. By using a mouse-adapted virulent CVB4 strain, Ramsingh and co-workers correlated two sites in capsid proteins VP1 (Halim & Ramsingh, 2000) and VP4 (Ramsingh & Collins, 1995) with expression of pancreovirulence. Tam et al. (2003) mapped five different sites in the CVB1 genome that contribute differentially to skeletal muscle inflammation and hind-limb weakness in a mouse model of myositis or chronic inflammatory myopathy. Two reports (Dunn et al., 2000, 2003) mapped the murine myocarditic phenotype of CVB3 clinical isolates to a short RNA sequence within the 5' NTR, termed domain II or stem–loop II (SLII).

CVB3 strain GA causes neither pancreatitis nor myocarditis in mice, but nonetheless replicates well in both murine heart and pancreas tissues (Tracy et al., 2002). Here, we report the characterization of an infectious cDNA copy of the CVB3/GA genome. Progeny CVB3/GA replicates identically to parental CVB3/GA and to lower titres in different cell cultures than virulent CVB3 strains. CVB3/GA causes no pathological changes in murine pancreas or heart tissue and immunizes mice safely against disease induced by virulent CVB3 challenge. Computer-aided secondary analysis of the CVB3/GA SLII sequence reveals differences from those predicted for virulent strains.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and viruses.
CVB3/GA was isolated in 1956, passaged minimally in cell culture and stored at the California State Public Health Laboratories, Berkeley, CA, USA; this strain was the kind gift of Dr David Schnurr. CVB3/GA replication and its lack of pathogenicity have been described previously (Tracy et al., 2000, 2002). Parental CVB3/GA stock was inoculated onto HeLa cell monolayers at an m.o.i. of 10 TCID50 per cell to provide virus for isolation of viral RNA. Prior to isolation of RNA, this stock of CVB3/GA was assayed in mice and was shown to be non-virulent (data not shown). CVB3/28 and CVB3/M have been described to induce pancreatitis and myocarditis in mice (Lee et al., 1997; Tracy et al., 2002). Viruses were titrated on HeLa cell monolayers [expressed as TCID50 (ml culture medium)–1] (Tracy et al., 2000). Murine fetal heart fibroblasts (MFHF) are a primary culture derived from adult C3H/HeJ mouse hearts (Tu et al., 1995). Non-obese diabetic (NOD) mouse pancreatic fibroblasts (NPF-1) were derived from pancreata of 4–6-week-old female NOD mice (Taconic Laboratories) during isolation of murine pancreatic islets of Langerhans. All cultures were propagated as monolayers in minimal essential medium with 10 % FBS and 50 µg gentamicin ml–1 at 37 °C in an humidified 5 % CO2 : 95 % air environment.

Cloning CVB3/GA RNA as infectious cDNA.
CVB3/GA was concentrated through 30 % (w/v) sucrose in 1 M NaCl, 20 mM Tris/HCl (pH 7·5), by using a Beckman SW28.1 rotor at 25 000 r.p.m. at 8 °C for 16 h. RNA was purified by using TRIzol LS reagent (Invitrogen). For reverse transcription (RT), 2 µg CVB3/GA RNA was mixed in water with 82 ng primer (PolyT-ClaI; 5'-GACGATCGAT28) and heated to 90 °C for 5 min prior to assembling the 20 µl RT reaction, which contained viral RNA, primer, 50 mM Tris/HCl (pH 8·3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 1 mM dNTPs, 20 U RNasin (Promega) and 400 U Superscript II reverse transcriptase (Invitrogen). After heating at 42 °C for 1·5 h, then at 75 °C for 15 min, the reaction was diluted to 100 µl with sterile, deionized water. PCR was carried out in 50 µl reaction volumes, containing 20 µl diluted CVB3/GA cDNA, 1 µl Elongase polymerase mix (Invitrogen), 0·4 mM dNTPs, 82 ng each of PolyT-ClaI and the return primer (5GAS2; 5'-ATACCGCGGttaaaacagcctgtgggttG), 60 mM Tris/H2SO4 (pH 9·1), 18 mM (NH4)2SO4 and 2 mM MgSO4. Elongase is a mixture of two different polymerases; the number of units of enzyme activity is not provided by the supplier. Amplification cycles were: one cycle at 94, 55 and 68 °C for 1 min each, followed by 35 cycles of 94 °C for 40 s, 51 °C for 40 s and 68 °C for 7·5 min, terminating with 68 °C for 10 min. Amplified cDNA was isolated, digested with SacII and ClaI and ligated into SacII- and ClaI-digested pKSCMV vector DNA. To generate pKSCMV, the 1 kb PvuI–StuI fragment of pSVN (Chapman et al., 1994) containing the NotI and ClaI sites was ligated with the 1·4 kb PvuII–PvuI restriction fragment of pBluescriptII SK(+) (Stratagene). The plasmid was linearized with SspI and ligated with a cytomegalovirus (CMV) promoter sequence amplified from pcDNA3.1 (Invitrogen) with primers 5CMV (5'-GGGAATATTCTGCTTCGCGATGTACGGGCCAGAT) and CMV (5'-AAAGCGGCCGCCGCGGAATTTCGATAAGCCAGTAAGCAGTG); the latter adds a SacII site downstream of the CMV promoter. The resulting pKSCMV vector has SacII and ClaI sites following the CMV promoter.

No completely full-length clones were identified. Restriction mapping identified a clone that lacked the region between nt 4000 and 5000. By using an SspI restriction fragment encompassing nt 4014–5139 from another clone that contained the 3' half of the CVB3/GA genome, a full-length CVB3/GA genome was assembled. Plasmid DNA was transfected into HeLa cell monolayer cultures (by using Effectene transfection reagent; Qiagen). Virus progeny from transfection was inoculated onto fresh HeLa cultures to create a virus stock. Virus was titrated on HeLa cells (TCID50 ml–1) and stored frozen at –75 °C.

Sequence analysis.
Sequence information for the parental CVB3/GA strain and progeny CVB3/GA virus was derived by using RT-PCR-generated amplimers or, in the case of the cloned CVB3/GA genome, from plasmid DNA. Sequence analysis was performed by the in-house core facility or by using a Thermonuclease Sequenase radiolabelled terminator cycle sequencing kit (USB Corporation). Sequence data were analysed with VectorNTI Advance 9.0 (Invitrogen). SLII secondary structural modelling was performed with the Mfold web server, using default values for all options (http://www.bioinfo.rpi.edu/applications/mfold/) (Zuker, 2003), including a folding temperature of 37 °C, 1 M NaCl, no divalent ions and calculation of all structures within 5 % free energy of the most stable structure.

Single-step growth curves.
Kinetics of replication were analysed in cell culture as described previously (Tu et al., 1995). Cell cultures (5x104 cells per well in 24-well plates) were inoculated at an m.o.i. of 20 TCID50 per cell into 0·3 ml medium, washed three times after 1 h, then cells were provided with fresh medium. Cultures were harvested at the times shown by freezing. Virus was titrated on HeLa cell monolayers.

Mice.
Virulence phenotypes and replication of virus were assayed in male C3H/HeJ mice (4–6 weeks old; Jackson Laboratories) (Chapman et al., 1994; Tracy et al., 1992; Tu et al., 1995). Mice were maintained at five per cage and provided with water and food ad libitum. Mice were inoculated intraperitoneally with 0·1 ml sterile 100 mM NaCl containing 5x105 TCID50 units of virus from stocks previously titrated on HeLa cell monolayers (Tracy et al., 1992). Sera were frozen and tissues were divided for pathological examination by fixation in buffered 10 % formalin or by freezing under dry ice for subsequent determination of infectious virus titre.

Titre of infectious virus in mouse heart and pancreas.
Tissues were weighed, homogenized in complete cell-culture medium, frozen and thawed three times and titrated on HeLa cell monolayers (Tracy et al., 2002). Virus titre was expressed as TCID50 (g tissue)–1.

Measurement of neutralizing-antibody titres.
Sera were heated at 56 °C for 45 min, chilled, then centrifuged for 5 min. Neutralizing titres against CVB3 were determined in a cytopathic reduction assay, as described elsewhere (Coller et al., 1990).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Analysis of the infectious CVB3/GA genome
The CVB3/GA sequence revealed differences relative to published CVB3 genomic sequences. No variation between parental and progeny CVB3/GA sequences was observed when the parental CVB3/GA sequence was examined at each position at which the progeny CVB3/GA sequence varied from other CVB3 sequences.

There is 81–83 % nucleotide sequence identity between the CVB3/GA 5' NTR and other CVB3 strains, consistent with the findings of others (Romero et al., 1997). Sixty-three sites within the CVB3/GA 5' NTR varied relative to other CVB3 sequences of known cardio- and pancreovirulence phenotype (Table 1). Forty-five of these are within a large portion of the 5' NTR in which secondary structure has been predicted (nt 1–644; Zell et al., 1999), whilst 24 of the sites are part of base pairs that are predicted to exist in these structures (Zell et al., 1999). Only four changes disrupt predicted base pairing; the remaining 20 either have compensatory mutations that maintain base pairing or change G : C base pairs to G : U or vice versa.


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Table 1. Sites in the CVB3/GA 5' NTR that vary relative to CVB3 sequences of known virulence phenotype

The CVB3/GA 5' NTR (nt 1–743) was aligned with those from CVB3/CO, CVB3/DO, CVB3/GU, CVB3/OL, CVB3/RE, CVB3/ZU and CVB3/AS (Romero et al., 1997), CVB3/20 (Tracy et al., 1992), CVB3/28 (Tracy et al., 2002), CVB3/M1 (GenBank accession no. M33854) and CVB3/M (Lee et al., 1997). No sequence information is available for CVB3/CO, CVB3/DO, CVB3/GU, CVB3/OL, CVB3/RE, CVB3/ZU or CVB3/AS for nt 1–82.

 
Twelve amino acid variations in the CVB3/GA P1 (capsid protein) coding region sequence (defined by nt 744–3296) are unique among CVB3 strains with known cardiovirulent phenotype (Table 2). No differences were observed in the well-conserved VP4 protein. Four sites (aa 153M, 156T, 162A and 173H) are within the VP2 ‘puff’ region (Muckelbauer et al., 1995), a surface loop at the receptor-binding canyon south rim where receptor interactions are likely (He et al., 2001). One site (aa 64T) in VP3 is in the knob (Muckelbauer et al., 1995), an antigenic region in PV and human rhinovirus 14 (Minor et al., 1986; Sherry et al., 1986). Two sites (VP1 aa 7L and 8A) are present in the N-terminal region of VP1, for which the structure has not been defined.


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Table 2. Sites in the CVB3/GA amino acid sequence of the P1 (capsid protein) region that vary from sequences in other CVB3 strains of known virulence phenotype

The predicted amino acid sequence of CVB3/GA (aa 1–851) was aligned with those of CVB3/CO, CVB3/DO, CVB3/GU, CVB3/OL, CVB3/RE, CVB3/ZU, CVB3/AS, CVB3/20, CVB3/28, CVB3/M1 and CVB3/M; sites at which the CVB3/GA sequence differed from all the aligned sequences were noted. S, Surface residue; {alpha}, {alpha} helix; {beta}, {beta} strand in structure defined by Muckelbauer et al. (1995).

 
Amino acid sequences of the CVB3/GA non-structural proteins were compared to homologous sequences of other HEV-B isolates (BLAST; Altschul et al., 1990), as other non-cardiovirulent CVB3 isolates have not been sequenced in this region of the genome. Eight CVB3/GA sites differ from all other HEV-B sequences that are available in GenBank (Table 3). These variations occur in protease 2A (aa 147), protein 2B (aa 4 and 95), protein 3A (aa 50) and protein 3D (aa 32, 37, 78 and 354).


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Table 3. Sites in the amino acid sequence of the P2 and P3 regions of CVB3/GA that vary from enteroviruses of the HEV-B subgroup

Predicted amino acid sequence of CVB3/GA proteins 2A, B, C, 3AB, C and D were aligned with nearly 100 enterovirus B amino acid sequences identified by BLAST (Altschul et al., 1990). Sites at which the sequence of CVB3/GA varied from all enterovirus B sequences are noted. Numbers in parentheses following the nucleotide indicate the number of viral sequences with that nucleotide at that position.

 
Alignment of the CVB3/GA 3' NTR (98 nt long; nt 7302–7400) with other enterovirus sequences identified two sites at which CVB3/GA varies uniquely: 7319G (C or A in other enteroviruses) and 7390U (A or G in other enteroviruses). Neither variation is in a region that is predicted to be base-paired (Merkle et al., 2002), although other interactions cannot be ruled out: nt 7319G is in the loop forming the Z stem, which is unique to the enterovirus B subgroup, and 7390U is an intervening nucleotide between the X and kissing (K) domains (Merkle et al., 2002). However, when the CVB3/GA 3' NTR sequence was compared to the structure that was modelled on the CVB3/M1 genome (Merkle et al., 2002), only 7325A (a variation that is not unique to the CVB3/GA genome) was predicted to disrupt base pairing in the Z stem. This sequence variation, 7325A, has also been reported in a CVB5 (Zhang et al., 1993) and in a CVB4 (GenBank accession no. AF311939) genome.

The cis-acting replication element (CRE) of the CVB3/GA genome (predicted to be located at nt 4365–4423 by analogy to PV; Goodfellow et al., 2000) has two unique variations at this position (C4376U and U4415C). Both sites are within the predicted CRE stem region. Nucleotide 4376U is thus predicted to alter a C : G base pair to U : G, whilst 4415C disrupts the neighbouring base pair of the stem. However, 19–20 bp of the stem remain intact and the changes are not within the portion of the terminal stem and loop that are found to be essential for PV replication (Goodfellow et al., 2000, 2003).

Replication of progeny CVB3/GA in cell culture
We functionally tested the hypothesis that progeny CVB3/GA is biologically identical to the parental strain by comparing replication in HeLa (Fig. 1a), MFHF (Fig. 1b) and NPF-1 (Fig. 1c) cultures. Parental and progeny CVB3/GA replicated indistinguishably from each other and to identical titres. Parental CVB3/GA replicates to lower titres in mouse pancreas and heart than do virulent CVB3 strains (Tracy et al., 2000). The replication of progeny CVB3/GA was therefore compared to that of CVB3/28, a myocarditic strain, in HeLa, MFHF and NPF-1 cultures. CVB3/GA and CVB3/28 replicated with identical kinetics and to equivalent titres in HeLa cells (Fig. 2a). However, in MFHF (Fig. 2b) and NPF-1 (Fig. 2c) cultures, avirulent CVB3/GA replicated with approximately 1·5–3-fold slower rates and to tenfold lower titres than the virulent strain CVB3/28.



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Fig. 1. Progeny ({blacksquare}) and parental ({square}) CVB3/GA replicate with identical kinetics and to equal titres in cell culture. Replication rates were assayed on (a) HeLa, (b) MFHF and (c) NPF-1 cultures. Infections were carried out by using an m.o.i. of 20. Titres were assayed on HeLa cell monolayers.

 


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Fig. 2. CVB3/GA ({square}) replicates with slower kinetics and to lower titres than a virulent CVB3 strain (CVB3/28; {bullet}) in primary murine cell cultures. Replication rates were assayed on (a) HeLa, (b) MFHF and (c) NPF-1 cultures. Infections were carried out by using an m.o.i. of 20. Titres were assayed on HeLa cell monolayers.

 
Progeny CVB3/GA induces neither pancreatitis nor myocarditis in mice
Parental CVB3/GA is non-pathogenic for pancreas and heart (Tracy et al., 2000). To determine the progeny CVB3/GA virulence phenotype, groups of five mice were inoculated with either virus; controls were inoculated with saline only or with virulent CVB3/M. Parental CVB3/GA induced neither myocarditis (Fig. 3c) nor pancreatitis (Fig. 3d) in any mouse; tissues were indistinguishable from those of mock-infected mice (Fig. 3a, b). Similarly, progeny CVB3/GA induced neither myocarditis (Fig. 3e) nor pancreatitis (Fig. 3f) in any mouse. These results contrasted sharply with severe myocarditis (Fig. 3g) and pancreatitis (Fig. 3h) induced by a virulent strain (CVB3/M).



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Fig. 3. Progeny CVB3/GA does not induce pancreatitis or myocarditis in experimentally inoculated mice. Mice were mock-infected with saline (panels a, b) or inoculated with parental CVB3/GA (c, d), progeny CVB3/GA (e, f) or virulent CVB3/M (g, h). Mice were killed 10 days post-inoculation and hearts (panels a, c, e, g) or pancreata (b, d, f, h) were fixed in formalin, paraffin-embedded, sectioned at 6 µm thickness and stained with haemotoxylin and eosin. Original magnification, x200.

 
CVB3/GA inoculation provides complete protection from disease induced by challenge with a virulent CVB3 strain
We tested the hypothesis that CVB3/GA can immunize mice against disease (pancreatitis and myocarditis) induced by virulent CVB3 challenge. Groups of five mice were inoculated with CVB3/GA or saline at 4, and again at 6, weeks of age. At 8 weeks of age, CVB3/GA- and saline-inoculated mice were inoculated with virulent CVB3/M. All mice were killed 10 days later. Although all mice in each group appeared normal, saline–CVB3/M-inoculated mice appeared smaller. Weights of mice in the CVB3/GA–CVB3/M group were compared to those in the saline–CVB3/M group. Mice in the CVB3/GA–CVB3/M group averaged 25·5±0·5 g, the same as uninoculated control mice at the same age, whereas saline–CVB3/M mice averaged 19±1 g per mouse, or 23 % less on average. Heart and pancreas tissue from mice inoculated twice with CVB3/GA, then killed 2 weeks later, appeared normal (Fig. 4a, b), whereas heart and pancreas tissues from each of the saline–CVB3/M mice showed widespread pancreatitis (Fig. 4c) and myocarditis (Fig. 4d). CVB3/GA–CVB3/M mice showed no evidence of cardiac (Fig. 4e) or pancreatic (Fig. 4f) virulence; these tissues appeared in all mice to be indistinguishable from those of mice inoculated only with CVB3/GA (as in Fig. 4a, b) or mock-infected mice (Fig. 3a, b).



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Fig. 4. Inoculation of mice with CVB3/GA provides protection against myocarditis and pancreatitis induced by challenge with a virulent CVB3 strain. Mice inoculated twice with CVB3/GA at 4 and 6 weeks of age showed no evidence of myocarditis (a) or pancreatitis (b) when killed 10 days following the last inoculation. Mice inoculated twice with CVB3/GA as above or with saline were challenged at 8 weeks of age with virulent CVB3/M, then killed 10 days later. CVB3/M induced myocarditis (c) and pancreatitis (d) in all mock-infected mice, but mice that were previously inoculated with CVB3/GA, then challenged with CVB3/M, were protected from myocarditis (e) and pancreatitis (f). Tissues were fixed, stained and sectioned as described for Fig. 3. Original magnification, x100.

 
Sera were assayed for anti-CVB3 neutralizing-antibody titres. Of five mice that were inoculated twice with CVB3/GA and killed at 8 weeks, one showed no titre of anti-CVB3 neutralizing antibodies at 1 : 8, whereas two mice had titres at 1 : 8, one at 1 : 16 and another at 1 : 32. Sera from the CVB3/GA–CVB3/M mice were then assayed; titres ranged between 1 : 32 (two mice), 1 : 64 (two mice) and 1 : 128 (one mouse), consistent with both protective immunity established by CVB3/GA replication and a boost provided by exposure to the challenge CVB3/M strain.

Predicted secondary structure of the sequence for SLII in the CVB3 5' NTR is different from that predicted for myocarditic or pancreovirulent CVB3 strains
Previous work demonstrated that a primary genetic determinant of the CVB3 myocarditis phenotype maps within the 5' NTR (Dunn et al., 2000) and specifically to domain II (or SLII; Dunn et al., 2003); cardiovirulent and pancreovirulent CVB3 SLII sequences have been predicted to have different secondary structures (Dunn et al., 2003). The SLII sequences for CVB3/GA, CVB3/28, CVB3/AS and CVB3/CO (Tracy et al., 2000) were analysed by using the Mfold web server (Zuker, 2003); only the energetically most favourable SLII structure for each strain is shown in Fig. 5. The SLII structure (nt 88–182) for CVB3/GA has a predicted {Delta}G of –18·6 kcal mol–1 (–77·8 kJ mol–1) (Fig. 5a). The sequences and energies for the other strains are as follows: CVB3/28 [nt 88–182; {Delta}G=–22·9 kcal mol–1 (–421·4 kJ mol–1)] (Fig. 5b), CVB3/AS [nt 88–186; {Delta}G=–20·1 kcal mol–1 (–84·1 kJ mol–1)] (Fig. 5c) and CVB3/CO [nt 88–186; {Delta}G=–24·0 (–100·4 kJ mol–1)] (Fig. 5d). Cardiovirulent strains CVB3/28 and CVB3/AS showed similar folding (Fig. 5b, c), whereas the structure of the non-cardiovirulent strain CVB3/CO SLII (Fig. 5d) differed markedly. The predicted SLII structure for the non-cardiovirulent, non-pancreovirulent strain CVB3/GA (Fig. 5a) also displayed different loop structures. Nucleotide differences at nt 123, 129, 132, 137, 161 and 162 contributed to differences in structure between CVB3/28 and CVB3/GA, including U161C (Table 1), which is unique to CVB3/GA. However, all SLII structures demonstrated structural conservation in the apical stem–loop (nt 139–160; boxed in Fig. 5). Alignment of 50 HEV-B SLII sequences selected by BLAST (Altschul et al., 1990) with CVB3/GA nt 88–182 or CVB3/28 nt 88–181 demonstrated that this base-paired region is maintained by compensatory mutations.



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Fig. 5. Predicted secondary structure of SLII in the CVB3 5' NTR is different from those in virulent CVB3 strains. The following sequences for domain II (SLII) structural comparison were chosen by alignment: (a) CVB3/GA nt 88–182; (b) CVB3/28 nt 88–181 (Tracy et al., 2002); (c) CVB3/AS (GenBank accession no. AF169670, nt 1–94); and (d) CVB3/CO (GenBank accession no. AF169665, nt 1–99). SLII structures were determined by using the Mfold web server as described in Methods. Predicted free energies of folding for this region of CVB3/GA, CVB3/28, CVB3/AS and CVB3/CO are –18·6, –22·9, –20·1 and –24·0 kcal mol–1 (–77·8, –421·4, –84·1 and –100·4 kJ mol–1), respectively. Circles denote base pairing (G : C, G : U and A : U). The apical loop is shown boxed. Nucleotide numbering is based on GenBank accession no. M88483. Arrows indicate differences between the CVB3/GA and CVB3/28 SLII sequences that contribute to RNA folding differences.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
CVB, discovered in the mid-20th century (reviewed by Dalldorf, 1955), were soon shown to be linked aetiologically to myocarditis (De Jager & Van Creveld, 1956; Disney et al., 1953; Kibrick & Benirschke, 1958) and, thereafter, to pancreatitis (Arnesjo et al., 1976; Imrie et al., 1977; Kennedy et al., 1986; Ursing, 1973). It is clear from murine models of CVB-induced myocarditis (reviewed by de Verdugo et al., 1995; Huber, 1997) and pancreatitis (Gomez et al., 1991; Ramsingh, 1997; Tracy et al., 2000) that different strains of CVB exhibit different virulence phenotypes (Gomez et al., 1991; Tracy et al., 2000). Attention has focused on CVB strains that are cardiovirulent (i.e. that induce myocarditis and pancreatitis in mice), due to the clinical importance of human heart disease; infectious CVB3 cDNAs have been derived from virus strains that exhibit a cardiovirulent phenotype (Kandolf & Hofschneider, 1985; Knowlton et al., 1996; Lee et al., 1997; Tracy et al., 1992). CVB3 strains that induce myocarditis in mice are rare, whereas pancreovirulent strains appear to be common (Tracy et al., 2000). To begin to understand the molecular genetics that determine the expression of CVB virulence phenotypes, we cloned and characterized the CVB3/GA genome.

The CVB3/GA 5' NTR varies from other CVB3 5' NTR sequences at 63 sites. The fact that only four of these alter predicted base pairing of 5' NTR secondary structure (Zell et al., 1999) indicates that predicted base pairing is likely to be vital for 5' NTR function. One site, nt 161U, is in SLII, disrupts a base pair in the stem–loop structure and is close to the site of a revertant mutation in a chimeric virus in which the 5' NTR of Human echovirus 12 (ECV12) replaced the 5' NTR of a CVB3 genome (Bradrick et al., 2001). Another unique variation, nt 123G, and two variations that are found only in non-cardiovirulent CVB3 strains (nt 132G and 162A; Romero et al., 1997) are critical for difference in predicted SLII folds between CVB3/GA and CVB3/28 (Fig. 5). Indeed, nt 118–120, 122, 123 and 126 map to the 5' side of the of the predicted bubble (defined by nt 118–140 and 160–170; Fig. 5a) of the CVB3/28 SLII structure and are sites of unique variation in CVB3/GA (Fig. 5; Table 1). Chemical probing of CVB3/28 5' NTR RNA demonstrates that nt 118–128 are protected from modification; this indicates that the region, although predicted in both CVB3 strains to have single-stranded regions (Zell et al., 1999), is in fact not single-stranded (W. Tapprich & J. Bailey, personal communication); this region may be involved in interactions with RNA elsewhere, potentially through tertiary interactions. Nucleotide 301A disrupts base pairing at the base of loop a in stem–loop IV, which has been shown to bind poly(rC) binding protein 2 (PCBP2) (Gamarnik & Andino, 2000), an essential translation host factor (Blyn et al., 1997). Although PCBP2 binding to the PV loop a is less critical for translation than loop b binding (Gamarnik & Andino, 2000), alterations of this structure may contribute to destabilizing protein interactions with stem–loop IV. Finally, another pair of variations that are unique to CVB3/GA (at nt 283 and 403) disrupt a base pair within this domain.

Sites with variations that are unique to the CVB3/GA 5' NTR were also observed in regions that current folding models predict are not base-paired. These include nt 427C, at which all other CVB3 strains of known virulence phenotype encode U; revertants of ECV12 : CVB3 5' NTR chimeras show a C->U mutation at this site (Bradrick et al., 2001). Other sites are found in the intercalating pyrimidine-rich tract between domains I and II, in the large bubble region in SLII discussed above, in the loop of domain V, in the intervening sequence between domains V and VI and in the sequence between domain VI and the ORF initiation codon. The nt 88–104 pyrimidine-tract region is predicted to have base pairing from chemical probes (W. Tapprich & J. Bailey, personal communication); thus, variation in this region may have effects on higher-order structure. Variations in the loop of domain V are not in sites of primary attenuating mutations in the PV genome, although mutational analysis of domain V has demonstrated its importance to internal ribosome entry site (IRES) function (Racaniello, 1988). The CVB3 sequence intervening between domains V and VI has been suggested to base-pair with 18S rRNA (Yang et al., 2003). The CVB3/GA sequence in this region has a similar number of possible base pairs with human 18S RNA nt 1805–1835 (Le et al., 1992); the extent of variation seen in this region of the CVB3 5' NTR indicates that specific base pairs may not be essential for such an interaction, but only perhaps some minimal amount of base pairing. Although a functional role has been described for the spacer region between the IRES and the ORF by deletional analysis (Iizuka et al., 1991), the degree of variation in this region, both in CVB3 strains and other enteroviruses, indicates that this role may not be dependent upon primary structure and is not likely to affect replication greatly. Indeed, mutational analysis to demonstrate the role of ribosomal scanning in this region of the CVB3 5' NTR indicated that the primary structure was not critically important for translation unless stable secondary structures could be formed that might block scanning (Yang et al., 2003).

Twelve sites vary between the predicted amino acid sequence for the CVB3/GA capsid proteins and other CVB3 strains of known virulence phenotype (eight sites of which are predicted to be on the surface of the capsid; Table 2). Four of these sites are in the VP2 puff (Muckelbauer et al., 1995), which may play a structural role in the formation of the receptor-binding canyon; however, none of these maps to a site in which the coxsackievirus and adenovirus receptor (CAR) binds (He et al., 2001). The VP2 puff and the VP3 knob (Muckelbauer et al., 1995) are regions in which neutralizing immunogenic sites map in rhino- and polioviruses (Minor et al., 1986; Page et al., 1988; Sherry & Rueckert, 1985; Sherry et al., 1986). The VP2 residues aa 153 and 156 and VP3 aa 64 may be part of a neutralizing-antibody immunogenic site; peptides of the CVB3/H3 sequence have been shown to induce CVB3 neutralizing antibody in rabbits and a peptide containing aa 162 can generate a T-cell response in mice (Auvinen et al., 1993). The VP2 puff is the site of an attenuated CVB3 strain that was generated as an antibody-escape mutant (Knowlton et al., 1996). It has been suggested that D2165 in CVB3/H310A1 attenuates by altering the electrostatic interaction of K2165 and E28 or E29 of the CAR protein (He et al., 2001). A study of peptides derived from the VP1 sequence identified a CVB3-immune IgG-binding peptide at aa 1–15; this peptide also induces proliferation of T cells from CVB3-immune mice and generates a protective immune response in mice (Haarmann et al., 1994; Huber et al., 1993). Two sites of unique CVB3/GA variation in VP1, aa 7 and 8, are within this immunogenic region. Overall, 10 of 12 sites with unique CVB3/GA variation in the capsid are exposed at the surface and/or may be immunogenically important. The demonstration that CVB3/GA induces protective immunity in mice against disease caused by challenge with a different, virulent CVB3 strain, however, shows that the capsid amino acid differences in CVB3/GA do not affect the generation of CVB3 type-specific immunity.

Eight positions in CVB3/GA non-structural proteins differ from all other enterovirus B sequences available in GenBank (Table 3). One site (aa 147) is in a C-terminal region of protein 2A that is essential for virus replication, but not for protease activity (Li et al., 2001); however, the alanine to valine change is a conservative substitution, making this substitution less likely to affect virus replication. Two differences occur in protein 2B at aa 4 and 95; neither site is in a region that is critical for the membrane-permeabilization function of this protein. However, others have shown that an insertion mutation at aa 5 prevents virus replication, whilst another insertional mutation, at aa 94, decreases virus yield (de Jong et al., 2004). CVB3/GA replicates well in cell culture and in mice, although to yields lower than those generated by virulent CVB3 strains (Drescher et al., 2004; Tracy et al., 2000, 2002); it is unlikely these two differences have significant impact upon CVB3/GA biology. The single site in protein 3A is not in the hydrophobic region that is essential for membrane association (Towner et al., 1996), nor is it in a dimerization region that is found in this protein (Strauss et al., 2003). Four sites were identified in the RNA-dependent RNA polymerase, protein 3D: aa 32 and 37 are in the N-terminal strand of the thumb domain, aa 78 is in the {alpha}A helix beneath the fingers domain and aa 354 is in the {beta}4 strand of the palm, by comparison to the structure of the PV polymerase (Hansen et al., 1997). The aa 32Y variation is also found in PV1, but substitution of this local region of CVB3 into the PV1 genome resulted in a RNA synthesis-defective phenotype (Cornell et al., 2004), showing that other residues are tuned uniquely to their own respective proteins. Residues aa 32Y, 37S and 354T are observed in enteroviruses outside the B subgroup, indicating that these variations alone may be relatively functional and may not decrease function. Clearly, the synergy of an entire protein sequence in creating functional efficiency is of prime importance; individual variations that also occur in the same proteins of other viruses may merely represent acceptable variation within the viral mutant swarm.

Biological characteristics of the progeny CVB3/GA strain were compared to those of the parental strain; these results also demonstrated that progeny CVB3/GA is an accurate biological reflection of the parental virus strain. Progeny virus replication was identical to that of the parental strain in different cell cultures and, when examined for pathogenicity in mice, demonstrated an inability to induce myocarditis and pancreatitis. This has also been well-documented for the parental strain in previous work (Tracy et al., 2000, 2002): CVB3/GA induces no disease in these tissues, despite replicating in pancreas and heart tissues of experimentally infected mice. Extending these data, we have now shown that CVB3/GA establishes protective anti-CVB3 immunity that prevents both pancreatitis and myocarditis upon subsequent challenge by a virulent strain of CVB3. These results demonstrate that CVB3/GA can be used safely to establish fully protective immunity against CVB3-induced disease in mice. With further characterization, CVB3/GA therefore could be considered to be a naturally occurring, vaccine-like CVB3 strain.

As CVB3/GA appears to be at one end of the CVB virulence spectrum, it represents a unique tool with which to explore the naturally occurring molecular genetics that control the expression of virulent CVB phenotypes in mouse models. Depredations of annual PV-induced poliomyelitis epidemics in the 20th century (Nathanson & Martin, 1979; Nathanson et al., 1993) prompted the creation of safe and efficient PV vaccines (Sabin, 1955; Sabin & Boulger, 1973; reviewed recently by Minor, 2003) that have eradicated poliomyelitis from most of the world. Despite this success, the molecular genetics that naturally caused the phenotype of a highly neurovirulent PV strain (Sabin, 1955) were not determined, although much is understood regarding attenuating mechanisms at work in Sabin PV vaccine strains (Evans et al., 1985; Kawamura et al., 1989; Kohara et al., 1988; Macadam et al., 1992, 1993; reviewed by Minor, 1992; Racaniello, 1988; Racaniello & Ren, 1996).

Several molecular approaches to attenuating disease-causing CVB phenotypes have been described (Chapman et al., 2000b; Gauntt et al., 1983; Ramsingh & Collins, 1995; Ramsingh et al., 1990; Tu et al., 1995; Willian et al., 2000), but relatively little is known about the molecular genetics that determine natural CVB virulence (or pathogenic) phenotypes. It is apparent from other models of human CVB-induced disease (Halim & Ramsingh, 2000; Kanno et al., 2001; Tam et al., 2003) that the induction of specific diseases can be affected by different regions of the CVB genome and that some of these appear to act in concert. SLII is a primary determinant of CVB-induced murine heart disease (Dunn et al., 2003); replacement of SLII in the genome of a virulent CVB3 strain with the SLII from a non-myocarditic CVB3 strain results in abrogation of the myocarditic phenotype, whilst replacement of the SLII from the same or different virulent CVB3 strain re-established the myocarditic phenotype. Comparison of the predicted secondary structures of SLII sequences from cardiovirulent and non-cardiovirulent CVB3 strains has shown that significant structural differences may exist (Dunn et al., 2003). It is unclear at present what the impact of nearly 10 % difference in 5' NTR nucleotide sequence and a different predicted SLII secondary structure may have upon determining the CVB3/GA virulence phenotype. The SLII sequences of the two virulent strains, CVB3/AS and CVB3/28, are highly similar, despite 15 years or more between their times of isolation. Similar sequences would be expected to fold similarly unless minor sequence differences have a profound effect upon high-order structure; this does not seem to be the case, as the predicted secondary structures are also very similar. Available 5' NTR sequences are all from CVB3 strains that were isolated in the early 1950s. It will be necessary to examine more SLII sequences from virulent CVB3 strains that have been isolated over many years to discriminate between two possibilities: (i) that the observed secondary structures reported here are closely dependent upon conservation of sequence, implying constraints upon movement of this sequence within sequence space, or (ii) that different sequences will fold like those of CVB3/AS and CVB3/28. Preliminary data from intratypic chimeric CVB3 strains in which the 5' NTRs have been switched between avirulent CVB3/GA and virulent CVB3/28 suggest that the CVB3/GA 5' NTR in the CVB3/28 background ablates myocarditis, whilst permitting pancreatitis to occur (K. Kono, unpublished results). Of note, replacing just the CVB3/28 SLII with that from CVB3/GA accomplished the same result (K. Kono, unpublished results). These data, together with results presented here and elsewhere (Dunn et al., 2000, 2003), strongly implicate SLII structure as the key determinant of myocarditis, but suggest that other regions influence pancreatitis induction.


   ACKNOWLEDGEMENTS
 
We thank William Tapprich and Jennifer Bailey for communicating unpublished results to us. We thank J. Smith Leser, Josilyn Butler and Prajoel Karki for excellent technical assistance. This work was supported in part by grants from the National Institutes of Health (N. M. C., K. M. D., S. T.), the Juvenile Diabetes Research Foundation (S. T.), the American Diabetes Association (S. T.) and the National Multiple Sclerosis Foundation (K. M. D.).


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 6 July 2004; accepted 22 September 2004.



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