Vol. 65, No. 3

JOURNAL OF VIROLOGY, Mar. 1991, p. 1286-1290 0022-538X/91/031286-05$02.00/0 Copyright ©3 1991, American Society for Microbiology

Selection of an Attenuated Coxsackievirus B3 Variant, Using a Monoclonal Antibody Reactive to Myocyte Antigen NANCY VAN HOUTEN, PATRICIA E. BOUCHARD, ALBERT MORASKA, AND SALLYI. HUBER* Department of Pathology, University of Vermont, Burlington, Vermont 05405-0068 Received 13 September 1990/Accepted 28 November 1990

Previously, we described a heart-reactive monoclonal antibody (MAb), lOAl, derived from a coxsackievirus B3 (CVB3)-infected mouse. This MAb selectively inhibits infection of HeLa cells and myocytes with the myocarditic virus variant (CVB3W). A plaque-purified variant (H3) of CVB3W was isolated from the heart of an infected animal, and a second virus (H3-1OAl) was obtained by growing H3 in HeLa cells in the presence of MAb lOAl. As with the parental CVB3W virus, H3 infection of HeLa cells can be inhibited by MAb lOA1, but the antibody-selected H3-1OA1 variant is resistant to MAb inhibition (presumably an escape mutant). BALB/c mice infected with 106 PFU of CVB3W, H3, or H3-1OA1 resulted in approximately 90% animal mortality with CVB3W or H3 and less than 10% mortality with H3-10Al, suggesting that the escape mutant is less pathogenic. Additionally, hearts from animals infected with H3-10A1 demonstrated only half the amount of myocarditis observed in either CVB3W- or H3-infected mice. Cardiac virus titers were also reduced approximately 200-fold in H3-10A1-infected animals compared with those in mice given the pathogenic variants. In vitro studies indicate that H3-1OA1 is less effective in inhibiting cellular RNA and protein synthesis and show reduced virus replication compared with that of pathogenic viruses in cultured myocytes.

Numerous factors influence viral pathogenicity, including genetic characteristics of the virus. Several wild-type coxsackivirus B3 (CVB3) variants have been described that differ dramatically in their capacity to induce myocarditis. In certain cases, CVB3 variants show minimal infectivity for myocardial cells in vitro and lack tropism for the heart in vivo (4). Failure of the virus to infect the heart undoubtably explains its poor pathogenicity for this organ. In other instances, some cardiotropic CVB3 variants infect the myocardium, initiate autoimmune responses to myocardial antigens, and trigger severe immunologically mediated myocarditis (myocarditic CVB3 [6, 13, 14]). However, not all cardiotropic CVB3 variants necessarily cause significant tissue injury. At least one wild-type CVB3 variant infects the myocardium but fails to initiate autoimmunity and produces minimal myocarditis (nonmyocarditic CVB3 [2, 5]). Cardiac virus concentrations are often slightly reduced in animals infected with nonmyocarditic viruses compared with those in animals infected with myocarditic CVB3, suggesting that pathogenicity of CVB3 variants might be associated with differences in virus replication within the cell. A major problem with evaluations of pathogenicity between these "wild-type" viruses, however, is the mutability of picornaviruses as a whole (10, 11) and the more than 20-year period during which these "wild-type'" CVB3 (Nancy) variants have been maintained separately. Undoubtably, many mutations have arisen, only some of which are relevant to the question of genetic factors in viral pathogenicity. Recently, we described a monoclonal antibody (MAb), MAb 10A, that distinguishes between the myocarditic and nonmyocarditic CVB3 variants (12). In the present communication, we extend this initial observation by showing that this MAb can be used to select a nonmyocarditic variant from a plaque-purified myocarditic CVB3 preparation. This escape mutant CVB3 variant also shows reduced replication in myocardial cells and is less effective than the wild type in *

inhibiting cellular metabolism in infected cells. These alterations in the infection process may control the pathogenic potential of CVB3. MATERIALS AND METHODS Animals. BALB/c mice were originally purchased from Cumberland Farms, Clinton, Tenn. Male mice 8 weeks of age and neonatal mice less than 72 h old were obtained from colonies of these mice maintained at the University of Vermont. Antibodies. Hybridoma clones 8A6 (CVB3 neutralizing antibody) and 1OAl were made as described in detail previously (12). Viruses. The wild-type CVB3 variant was originally obtained from the late Jack Woodruff (myocarditic CVB3, hereafter designated as the CVB3W variant). This virus was grown in HeLa cells, and titers were determined by the plaque forming assay on HeLa cell monolayers as described elsewhere (1). Two new variants of CVB3W were obtained as follows. An 8-week-old male DBA/2 mouse was inoculated intraperitoneally with 105 PFU of CVB3W and sacrificed 7 days later. The heart was removed, homogenized in 1 ml of Dulbecco modified essential medium (DMEM; GIBCO, Grand Island, N.Y.) containing 100 U of penicillin and 100 ,ug of streptomycin per ml and 5% fetal bovine serum (FBS; GIBCO), and centrifuged at 300 x g for 10 min to remove cellular debris. The supernatant was serially diluted in DMEM-5% FBS, and 0.2 ml was added to confluent HeLa cell monolayers in 60-mm tissue culture plates (Corning Glass Works, Corning, N.Y.). After incubation for 1 h at 370C, the monolayers were overlaid with medium containing 1% agar and incubated at 37°C for 2 days. The plates were examined with a inverted microscope for plates containing fewer than five plaques, and the agar over a single plaque was removed with a Pasteur pipette. The agar was homogenized in 1 ml of DMEM-5% FBS and plaque purified as described above; this resulted in the CVB3-H3 virus variant. Next, confluent flasks of HeLa cell monolayers in 25-cm2

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tissue culture flasks (Corning) were incubated for 1 h at 37°C with 100 ,ug of MAb lOA1; then 104 PFU of CVB3-H3 was added in 2 ml of DMEM-5% FBS, and the flasks were incubated overnight at 37°C in 6% CO2. Since the HeLa cells showed no cytopathic effect, the cells were detached with a rubber policeman and the supernatant and cells were sequentially frozen and thawed three times. Cellular debris was removed by centrifugation at 325 x g for 10 min, and 10% (final volume) cell lysate was added to fresh HeLa cell monolayers in the presence of 100 ,ug of MAb 1OAl. After an additional blind passage, the monolayers were incubated for 48 h at 37°C until the cytopathic effect was complete. The cells and supernatant were removed and alternately frozen and thawed three times, and the cell debris was removed by centrifugation at 325 x g for 10 min. This virus preparation was designated CVB3-H3-lOA1 (H3-IOA1). H3-1OA1 was titered by the plaque-forming assay and subjected to plaque purification as described above. Preparation of myocytes. Hearts were asceptically removed from neonatal BALB/c mice less than 72 h old, minced finely, subjected to stepwise enzymatic dissociation with 0.4% collagenase II, and cultured in DMEM containing antibiotics, 5% FBS, and 10% horse serum at a concentration of 3 x 105 viable cells per ml (12). This procedure results in a cell population containing more than 90% myocytes. Plaque forming assay. HeLa cell monolayers in 60-mm tissue culture plates were cultured with MAb or control antibody concentrations as stated above and approximately 100 PFU of virus in DMEM-5% FBS. After incubation for 45 min at 37°C, the monolayers were washed to remove unbound virus and antibody, overlaid with 5 ml of medium with 0.6% agar, and incubated for 48 h at 37°C in 5% CO2. The monolayers were fixed with 10% buffered Formalin and stained with 0.5% crystal violet. Kinetics of virus infection of myocytes. Myocytes (3 X 103) were cultured in tissue culture plates (1-mm-diameter wells) for 2 days, washed once with medium, and incubated with 3 x 105 PFU of virus at 37°C. At the times stated, 5 ,ug of CVB3 neutralizing antibody (500 [Lg/ml) was added to the wells for 15 min. The wells were washed three times with medium and cultured in DMEM-5% FBS at 370C for 6, 12, 24 h. The cells and supernatant from each well were retrieved, alternately frozen and thawed three times to maximally release virus, and titered by the plaque forming assay. [3H]uridine and [3H]leucine incorporation in infected myocytes. Myocytes (6 x 104) were cultured in tissue culture plates (6-mm-diameter wells) for 2 days, washed once with medium, and incubated for the times stated with 100 PFU of virus per cell. Control cultures were continued in medium without virus. All cultures received 2 p.Ci of either [5,6-3H] uridine (42 Ci/mM; ICN Pharmaceuticals) or L-[3,4,5-3H] leucine (147 Ci/mM; New England Nuclear Research Products, Boston, Mass.); they were incubated for an additional 2 h and then harvested onto glass filter strips by using a multiple automatic sample harvester (MASH; Belco Glass Inc.). The strips were air dried, and the sections containing radioactivity were placed in vials with 5 ml of Ecolite liquid scintillation fluid (Westchem, San Diego, Calif.) and counted in a Beckman liquid scintillation counter. Histology. Animals were sacrificed by lethal injection of sodium pentobarbital (Fort Dodge Laboratories, Inc., Fort Dodge, Iowa); hearts were removed, fixed in 10% buffered Formalin, sectioned, and stained with hematoxylin and eosin. Sections were scored blindly for myocarditis by S.A.H. using a scale of 0 to 4 as follows: 0, no myocarditis; 1, between 1 and 10 foci per section; 2, between 11 and 20

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foci per section; 3, between 21 and 40 foci per section; 4, widespread and/or confluent areas of inflammation (>20% of the myocardium inflamed as determined by image analysis studies). Statistical analysis. Statistical evaluations were performed by using either the Wilcoxon ranked score or the Student t test. RESULTS Selection of an attenuated CVB3 variant by using MAb lOAl. H3, a plaque variant of CVB3W, was derived from the heart of an infected mouse as described in Materials and Methods. H3-1OA1, a plaque variant of H3, was selected for its ability to infect HeLa cells in the presence of MAb lOAl and presumably represents an escape mutant virus. The following experiment demonstrates that the escape mutant virus is resistant to MAb lOAI inhibition of infection. Approximately 100 PFU of CVB3W, H3, or H3-1OA1 were incubated on HeLa cell monolayers in either medium alone or medium containing 1, 5, 10, or 20 p.g of MAb 8A6 (a CVB3 neutralizing antibody effective against all variants irrespective of pathogenicity), MAb lOAl, or normal mouse immunoglobulin per plate (Fig. 1). As expected, MAb 8A6 efficiently inhibited infection by all three virus variants. MAb lOAl, however, showed a dose-dependent inhibition of CVB3W and H3 but was ineffective against the escape mutant, H3-1OA1. Next, BALB/c mice were inoculated intraperitoneally with either 102, 104, or 106 PFU of these three viruses. Figure 2 demonstrates the cumulative mortality of 10 animals per group. This represents the results in one of two replicate experiments; both experiments gave identical results. CVB3W and H3 variants induced significant mortality, especially at the higher virus inocula. However, H3-1OA1 infections resulted in the death of only 1 of a total of 30 mice. Hearts from animals infected with 104 PFU of the three virus variants were subsequently evaluated for both myocarditis and virus concentrations 7 days after infection (Fig. 3). In two separate experiments, animals infected with H3-1OA1 showed significantly less myocarditis than did H3or CVB3W-infected mice. Generally, cardiac virus concentrations were also lower in H3-1OA1-infected mice, although considerable variability was observed. No correlation between myocarditis score and virus concentration was evident, however, when comparisons were performed between animals with equivalent virus titers in the three groups. That is, when H3-1OA1- and H3-infected animals were paired for equivalent levels of virus in the heart, H3-1OA1-infected mice continued to demonstrate less myocarditis than did the H3-infected mice. One question was whether the virus detected in the hearts of H3-1OA1-infected mice remained resistant to MAb inhibition or represented a natural reversion to the wild-type phenotype. Titers of heart homogenates from four to five mice infected with either CVB3W, H3, or H3-1OA1 were determined on HeLa cell monolayers in the presence of 18 1Lg of MAb lOAl or normal immunoglobulin by using the plaque forming assay. The percent inhibition of plaque formation in the presence of MAb lOAl was determined relative to that in the presence of control immunoglobulin. This study demonstrated that between 70 and 90% of PFU from CVB3W- and H3-infected mice could be inhibited by MAb lOA1, compared with 0 to 20% of virus from H3-1OA1-infected animals. Therefore, virus retrieved from H3-1OA1-infected animals continued to be resistant to MAb lOAl inhibition. Comparisons between CVB3W, H3, and H3-1OA1 infec-

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with 100 PFU/cell for 0, 1, 3, or 6 h and labeled with either [3H]uridine or [3H]leucine (Fig. 4). Both CVB3W and H3 infections reduced [3H]uridine incorporation by approximately 30% and [3H]leucine incorporation by approximately 35%. H3-1OA1 infections were less inhibitory, resulting in

tions. Possible reasons for the reduced H3-1OA1 virus concentrations in the heart were investigated. The initial study determined whether pathogenic and nonpathogenic variants showed equivalent abilities to interfere with cellular metabolism in infected cells. Myocyte monolayers were cultured

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VIRUS FIG. 3. Comparison of myocarditis and cardiac virus concentrations in mice inoculated with three CVB3 variants. Groups of 10 BALB/c mice were injected intraperitoneally with 104 PFU of CVB3W, H3, or H3-1OA1 and sacrificed 7 days later. Hearts were removed and divided in half. Half of each heart was fixed, sectioned, stained with hematoxylin and eosin, and scored for inflammation (a and c). Virus titers were determined in the remaining tissue by using the plaque forming assay (b and d). Results are given as mean myocarditis score per group and individual animal viral titers for two replicate experiments. *, Data are significantly different from those of CVB3W-infected mice (P c 0.005, Wilcoxon ranked score analysis). 3H-Uridine Incorporation

only 9 and 14% inhibition of uridine and leucine incorporation, respectively, at 6 h after infection. Next, myocytes were cultured with 100 PFU/cell for 0, 1, 2, 4, 10, 20, 40, 60, and 90 min, and extracellular virus was neutralized with 500 mg of MAb 8A6 per ml. The monolayers were washed and incubated for an additional 24 h. Virus production in the culture was determined by the plaque forming assay (Fig. 5). Generally, virus exposure times of more than 10 min were needed to consistently infect the cultures. No significant differences in time required for infection were observed for any of the three CVB3 variants. Both H3- and CVB3Winfected myocytes produced approximately 100-fold more virus per culture than did myocytes infected with H3-1OA1 virus, however, suggesting that the decreased cardiac titersin vivo may reflect poorer H3-1OA1 replication and/or release in myocytes rather than differences in infectivity of the different viruses in these cells.

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Picornaviruses are highly mutable viruses, and variants of the same virus can differ dramatically in their pathogenic potential (3, 4, 10, 11). Several distinct characteristics of either the virus or infection process might influence pathogenicity. Certainly, tropism of a virus for a particular tissue can affect the ability of the virus to induce damage to that tissue (3). However, virus tropism does not completely explain the poor pathogenicity of H3-1OA1 virus. Cardiac virus titers are lower in H3-1OA1-infected animals compared with those in mice given either H3 or CVB3W. However, the range in cardiac virus titers among all three groups of

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infected animals is wide, and the titers of a proportion of H3-1OA1-infected mice are equal to those of a proportion of H3- and CVB3W-infected mice. Yet when comparisons are performed between CVB3W-, H3-, and H3-1OAI-infected mice with equal virus concentrations in the heart, the H3-1OA1-infected mice still do not show as much myocarditis as the pathogenic virus-infected individuals. Picornaviruses frequently interfere with normal cellular metabolism (7). Such an effect in a metabolically active cell could prove to be highly detrimental to its function and may actually provide the foundation for pathogenesis (8). Specific mutations at the noncoding 5' end of the encephalomyocarditis virus seem to play an essential role in the pathogenicity of this virus and may mediate this effect through alterations in either virus infection or biological function (cytokine production) of macrophages (9). In the present study, we have described an MAb that presumably detects relevant differences in CVB3 infections that are influential in determining virus pathogenicity. When the H3-1OA1 variant was isolated from the (myocarditic) H3 virus preparation by using the MAb, the escape mutant demonstrated dramatically less pathogenicity both by animal mortality and by myocarditis induction. Since H3 was derived by plaque assay from CVB3W, this preparation probably is relatively homogenous compared with that of the parent virus. Therefore, despite the high mutability of picornaviruses, presumably fewer irrelevant genetic alterations will exist between H3 and H3-1OA1 than between the wild-type variants, which have been separated for many years. Interestingly, although the parental and escape mutants both show equivalent abilities to infect (i.e., penetrate) cultured myocytes, the nonpathogenic variant is less effective in inhibiting cellular metabolism and producing progeny virions in infected cells. Possibly, any decreased ability in converting cellular metabolism to viral replication would explain poorer virus yields. Whether this fact also explains the poorer pathogenicity of

ACKNOWLEDGMENTS We thank Laurie Sabens for expert secretarial assistance and Judy Kessler for preparation of the illustrations. This work was supported by Public Health Service grants 5-POlA126367 and HL28833 from the National Institutes of Health and by American Heart Association grant 90-999. N.V.H. was supported by National Institutes of Health Environmental Pathology Training Grant PHS T32 07122-08. REFERENCES 1. Blay, R., K. Simpson, K. Leslie, and S. Huber. 1989. Coxsackievirus-induced disease. CD4+ cells initiate both myocarditis and pancreatitis in DBA/2 mice. Am. J. Pathol. 135:899-907. 2. Gauntt, C. J., M. D. Trousdale, D. R. L. LaBadie, R. E. Paque, and T. Nealon. 1979. Properties of coxsackievirus B3 variants which are amyocarditic or myocarditic for mice. J. Med. Virol. 3:207-220. 3. Girard, M., A. Martin, T. Couderc, R. Crainic, and C. Wychowski. 1989. Modification of six amino acids in the VP1 capsid protein of poliovirus type 1, Mahoney strain, alters its host range and makes it neurovirulent for mice, p. 265-279. In B. L. Semler and E. Ehrenfeld (ed.), Molecular aspects of picornavirus infection and detection. American Society for Microbiology, Washington, D.C. 4. Huber, S. A., C. Haisch, and P. A. Lodge. 1990. Functional diversity in vascular endothelial cells: role in coxsackievirus tropism. J. Virol. 64:4516-4522. 5. Huber, S. A., and L. P. Job. 1983. Differences in cytolytic T-cell response of BALB/c mice infected with myocarditic and nonmyocarditic strains of coxsackievirus type B, type 3. Infect. Immun. 39:1419-1427. 6. Huber, S. A., and P. A. Lodge. 1984. Coxsackievirus B3 myocarditis in Balb/c mice. Evidence for autoimmunity to myocyte antigens. Am. J. Pathol. 116:21-29. 7. Jen, G., B. M. Detjen, and R. E. Thack. 1980. Shutoff of HeLA cell protein synthesis by encephalomyocarditis virus and poliovirus: a comparative study. J. Virol. 35:150-156. 8. Oldstone, M. B. A., M. Rodriguez, W. H. Daughaday, and P. W. Lampert. 1984. Viral perturbation of endocrine function: disordered cell function leads to disturbed homeostasis and disease. Nature (London) 307:278-281. 9. Palmenberg, A. Personal communication. 10. Prabhakar, B. S., M. A. Menegus, and A. L. Notkins. 1985. Detection of conserved and nonconserved epitopes on coxsackievirus B4: frequency of antigenic change. Virology 146:302316. 11. Tokskaya, E. A., L. I. Romanova, V. M. Blinov, E. G. Viktorova, A. N. Sinyakov, M. S. Kolesnikova, and V. I. Agol. 1987. Studies on the recombination between RNA genomes of poliovirus: the primary structure and nonrandom distribution of crossover regions in the genomes of intertypic poliovirus recombinants. Virology 161:54-61. 12. Weller, A. H., K. Simpson, M. Herzum, N. Van Houten, and S. A. Huber. 1989. Coxsackievirus B3-induced myocarditis virus receptor antibodies modulate myocarditis. J. Immunol. 143:1843-1850. 13. Wolfgram, L. J., K. W. Beisel, A. Herskowitz, and N. R. Rose. 1986. Variations in the susceptibility to coxsackievirus B3induced myocarditis among different strains of mice. J. Immunol. 136:1846-1852. 14. Woodruff, J. E., and J. J. Woodruff. 1974. Involvement of T lymphocytes in the pathogenesis of Coxsackie virus B3 heart disease. J. Immunol. 113:1726-1734.

Selection of an attenuated Coxsackievirus B3 variant, using a monoclonal antibody reactive to myocyte antigen.

Previously, we described a heart-reactive monoclonal antibody (MAb), 10A1, derived from a coxsackievirus B3 (CVB3)-infected mouse. This MAb selectivel...
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