Murine natural killer cells limit coxsackievirus B3 replication

Previous indirect evidence suggested that natural killer (NK) cells play a role in coxsackie virus B3 serotype 3, myocarditic variant (CVB3m)-induced myocarditis by limiting virus replication. In this study, we present direct evidence that NK cells can limit CVB3m replication both in vitro and in vivo. Virus titers are lowered in primary murine neonatal skin fibroblast (MNSF) cultures incubated with activated splenic large granular lymphocytes (LGL) taken from mice 3 days postinoculation of CVB3m, a time of maximal NK cell activity. The antiviral effect of this cell population is diminished by complement-mediated lysis with the use of anti-asialo GM1 antiserum but not with anti-Lyt-2 monoclonal antibody. Neither interferon nor anti-CVB3m-neutralizing antibody was detected in these cultures. Although activated LGL initiate lysis within CVB3m-infected MNSF in vitro within 3 hr of addition, they do not lyse uninfected MNSF cultures. CVB3m replication is required for expression of surface changes on MNSF that result in lysis by NK cells because cell cultures treated with compounds that prevent CVB3m replication are not killed by LGL. LGL also do not lyse MNSF cultures inoculated with UV-inactivated virus. Mice inoculated with activated LGL and subsequently challenged with CVB3m had reduced titers of virus in heart tissues in comparison to titers of CVB3m in heart tissues of mice not given LGL. The antiviral activity of the LGL preparation was abolished by prior treatment with anti-asialo GM1 antiserum plus complement but not by prior treatment with anti-Lyt-2 monoclonal antibody and complement. These data suggest that NK cells can specifically limit a nonenveloped virus infection by killing virus-infected cells.     

Involvement of natural killer cells in coxsackievirus B3-induced murine myocarditis

The role of natural killer cells in the temporal development of coxsackievirus B3-induced myocarditis in adolescent CD-1 male mice was examined. Inoculation of purified CVB3m induced maximum NK cell activity in the splenic populations at 3 days postinoculation (p.i.) as assessed by lysis of YAC-1 cells; maximum virus titers in heart tissues were also found at day 3 p.i. Mice depleted of NK cells after injection of anti-asialo GM1 antiserum i.v. had decreased NK cell activity, increased CVB3m titers in heart tissues, and exacerbated myocarditis. Although lesion number was not increased in heart tissues of the latter mice, lesions in these mice exhibited increased myocyte degeneration and dystrophic calcification above that found in lesions of mice inoculated with CVB3m only. No alteration in interferon titers were observed in CVB3m-infected mice treated with anti-asialo GM1 antiserum as compared with normal CVB3m-infected mice. Measurements of splenic NK cell activity in mice inoculated with doses of 10(2) to 10(8) PFU of CVB3m per mouse or UV-irradiated virus suggest that replication of CVB3m is required for NK cell activation. An amyocarditic variant of CVB3m (ts5R) was shown to replicate in heart tissues and to elicit NK cell activity comparable to that elicited by CVB3m. Therefore, the data suggest that NK cell activation depends on virus replication and that these cells provide some protection against CVB3m-induced myocarditis by limiting virus replication in heart tissues.     

Cardiac injury in myocarditis induced by Coxsackievirus group B, type 3 in Balb/c mice is mediated by Lyt 2 + cytolytic lymphocytes

Male Balb/c mice inoculated with a heart-adapted variant of Coxsackievirus, group B, type 3 (CVB3) develop severe myocarditis 7 days later. The lesions are characterized by mononuclear cell inflammation and myocyte necrosis. Infected T-lymphocyte-deficient mice show either minimal or no cardiac injury, although virus concentrations in the hearts of T-cell-deficient and -sufficient animals are similar. Adoptive transfer of 2 X 10(6) CVB3 immune Thy 1+ cells into CVB3-infected T-cell-deficient mice effectively restored myocarditis to levels observed in intact animals. Similar reconstitution with immune Ig+ cells or serum resulted in only a minimal increase in cardiac injury. To determine whether T-lymphocyte-dependent humoral or cellular immunity was responsible for myocarditis. T lymphocytes were obtained from Balb/c mice 6 days after infection with CVB3, separated into Lyt 1+2- (helper) and Lyt 1-2+ (cytolytic/suppressor) cell populations, and 2 X 10(6) of the enriched helper and cytolytic cells were adoptively transfused into infected T-cell-deficient recipients. Animals receiving the immune Lyt2+ cells developed severe myocarditis, had cytolytic T lymphocytes to both CVB3-infected and uninfected myocytes, but lacked a detectable IgG antibody response. Recipients of the Lyt 1+ cells failed to develop either myocarditis or cytolytic T cells but had normal serum IgG antibody titers to the virus. These results demonstrate that cardiac myocarditis is the product of cellular immune mechanisms.     

Murine cell-mediated immune response recognizes an enterovirus group-specific antigen(s).

Splenocytes taken from mice inoculated with coxsackievirus B3 (CVB3) (Nancy) developed an in vitro proliferative response against CVB3 antigen. This response could not be detected earlier than 8 days postinoculation but could be detected up to 28 days after exposure to CB3. CVB3-sensitized splenocytes responded not only to the CVB3 antigen but to other enteroviruses as well. This response was found to be enterovirus specific in that no response was detected to a non-enteroviral picornavirus, encephalomyocarditis virus, or to an unrelated influenza virus. The generation of a splenocyte population capable of responding to an enterovirus group antigen(s) was not limited to inoculation of mice with CVB3, as similar responses were generated when mice were inoculated with CVB2. Cell subset depletions revealed that the major cell type responding to the enterovirus group antigen(s) was the CD4+ T cell. Current evidence suggests that the group antigen(s) resides in the structural proteins of the virus, since spleen cells from mice inoculated with a UV-inactivated, highly purified preparation of CVB3 virions also responded in vitro against enteroviral antigens.     

Using recombinant coxsackievirus B3 to evaluate the induction and protective efficacy of CD8+ T cells during picornavirus infection

Coxsackievirus B3 (CVB3) is a common human pathogen that has been associated with serious diseases including myocarditis and pancreatitis. To better understand the effect of cytotoxic T-lymphocyte (CTL) responses in controlling CVB3 infection, we have inserted well-characterized CTL epitopes into the CVB3 genome. Constructs were made by placing the epitope of interest upstream of the open reading frame encoding the CVB3 polyprotein, separated by a poly-glycine linker and an artificial 3Cpro/3CDpro cleavage site. This strategy results in the foreign protein being translated at the amino- terminus of the viral polyprotein, from which it is cleaved prior to viral assembly. In this study, we cloned major histocompatibility complex class I-restricted CTL epitopes from lymphocytic choriomeningitis virus (LCMV) into recombinant CVB3 (rCVB3). In vitro, rCVB3 growth kinetics showed a 1- to 2-h lag period before exponential growth was initiated, and peak titers were approximately 1 log unit lower than for wild-type virus. rCVB3 replicated to high titers in vivo and caused severe pancreatitis but minimal myocarditis. Despite the high virus titers, rCVB3 infection of naive mice failed to induce a strong CD8+ T-cell response to the encoded epitope; this has implications for the proposed role of "cross-priming" during virus infection and for the utility of recombinant picornaviruses as vaccine vectors. In contrast, rCVB3 infection of LCMV-immune mice resulted in direct ex vivo cytotoxic activity against target cells coated with the epitope peptide, demonstrating that the rCVB3-encoded LCMV-specific epitope was expressed and presented in vivo. The preexisting CD8+ memory T cells could limit rCVB replication; compared to naive mice, infection of LCMV-immune mice with rCVB3 resulted in approximately 50-fold-lower virus titers in the heart and approximately 6-fold-lower virus titers in the pancreas. Although the inserted CTL epitope was retained by rCVB3 through several passages in tissue culture, it was lost in an organ-specific manner in vivo; a substantial proportion of viruses from the pancreas retained the insert, compared to only 0 to 1.8% of myocardial viruses. Together, these results show that expression of heterologous viral proteins by recombinant CVB3 provides a useful model for determining the mechanisms underlying the immune response to this viral pathogen.     

Aggravation of coxsackievirus, group B, type 3-induced myocarditis and increase in cellular immunity to myocyte antigens in pregnant Balb/c mice and animals treated with progesterone

Male Balb/c mice inoculated with a heart-adapted variant of coxsackievirus, group B, type 3 (CVB3M) develop severe myocarditis characterized by extensive focal lesions of inflammatory cells and necrosis of the myocardium. Females generally develop minimal myocarditis except when infected during the first and third trimesters of pregnancy. Enhanced myocarditis is usually accompanied by elevations in virus concentrations in the heart, virus-specific antibody titers, and lymphocyte mediated cytolytic activity to both uninfected and CVB3M-infected myocytes in vitro. As previously shown in males, T-lymphocyte-depleted pregnant female mice inoculated with the virus do not develop significant myocarditis indicating that immune rather than virus-mediated myocyte damage is important in myocarditis. Progesterone increases during gestation reaching maximum concentrations during the third week when heart disease is most severe. Administration of progesterone to castrated male and female mice prior to virus inoculation resulted in increased virus concentrations, cellular and humoral CVB3M-specific immunity, and myocarditis. Two hypotheses for exacerbation of the disease with elevated progesterone concentrations have been postulated: the hormone either indirectly increases cellular immune responses by enhancing virus replication, or independently enhances both T-cell responses and virus replication.     

Interacting nutritional and infectious etiologies of Keshan disease

In 1979, Chinese scientists reported that selenium had been linked to Keshan disease, an endemic juvenile cardiomyopathy found in China. However, certain epidemiological features of the disease could not be explained solely on the basis of inadequate selenium nutrition. Fluctuations in the seasonal incidence of the disease suggested involvement of an infectious agent. Indeed, a coxsackievirus B4 isolated from a Keshan disease victim caused more heart muscle damage when inoculated into selenium-deficient mice than when given to selenium-adequate mice. Those results led us to study the relationship of nutritional status to viral virulence. Coxsackievirus B3/0 (CVB3/0), did not cause disease when inoculated into mice fed adequate levels of Se and vitamin E. However, mice fed diets deficient in either Se or vitamin E developed heart lesions when infected with CVB3/0. To determine if the change in viral phenotype was maintained, we passaged virus isolated from Se-deficient hosts, maintained, we passaged virus isolated from Se-deficient hosts, designated as CVB3/0 Se-, back into Se-adequate hosts. The CVB3/0 Se- virus caused disease in Se-adequate mice. To determine if the phenotype change was due to changes in the viral genome, we sequenced viruses isolated from Se-deficient mice and compared them with the input CVB3/0 virus. Six point mutations differed between the parent strain and the recovered CVB3/0 Se- isolates. When the experiment was repeated using vitamin E-deficient mice, the same 6 point mutations were found. This is the first report of a specific host nutritional deficiency altering viral genotype. Keshan disease may be the result of several interacting causes including a dominant nutritional deficiency (selenium), other nutritional factors (vitamin E, polyunsaturated fatty acids), and an infectious agent (virus).     

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 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 (H3-10A1) was obtained by growing H3 in HeLa cells in the presence of MAb 10A1. As with the parental CVB3W virus, H3 infection of HeLa cells can be inhibited by MAb 10A1, but the antibody-selected H3-10A1 variant is resistant to MAb inhibition (presumably an escape mutant). BALB/c mice infected with 10(6) PFU of CVB3W, H3, or H3-10A1 resulted in approximately 90% animal mortality with CVB3W or H3 and less than 10% mortality with H3-10A1, 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 H3-10A1 is less effective in inhibiting cellular RNA and protein synthesis and show reduced virus replication compared with that of pathogenic viruses in cultured myocytes.     

Benign coxsackievirus damages heart muscle in iron-loaded vitamin E-deficient mice

Several oxidative stressors (dietary selenium deficiency, dietary vitamin E deficiency coupled with fish oil feeding, genetic reduction of glutathione peroxidase activity) allow a normally benign coxsackievirus B3 (CVB3/0) to damage heart muscle in host mice. This study investigated whether dietary iron overload, another oxidant stress, would also permit CVB3/0 to exert a cardiopathologic effect in vitamin E-deficient (-VE) mice. Four groups of mice were fed either a -VE or a +VE diet containing either an adequate or an excessive (30x) amount of iron. After 4 weeks of feeding, the mice were inoculated with CVB3/0 and heart damage was assessed at various times postinfection. Mice fed a diet sufficient in VE with excess iron developed heart damage equivalent to mice fed a diet deficient in vitamin E without excess iron. However, severe heart damage occurred in the group fed a diet deficient in VE with excess iron, which was the most pro-oxidative diet. The highest heart viral titers were found in mice fed the -VE/excessive iron diet. However, the extent of heart damage did not always correlate with the formation of TBARS in liver homogenates. Further research is needed to clarify the role of oxidative stress and iron overload in determining the course of viral infection.     

Increased fatigue of isovelocity vs. isometric contractions of canine diaphragm

A comparison of fatigue as a loss of force with repeated contractions over time was performed in canine respiratory muscle by isometric (nonshortening) and isovelocity (shortening) contractions. In situ diaphragm muscle strips were attached to a linear ergometer and electrically stimulated (30 or 40 Hz) via the left phrenic nerve to produce either isometric (n = 12) or isovelocity (n = 12) contractions (1.5 s) from optimal muscle length (Lo = 8.8 cm). Similar velocities of shortening between isovelocity experiments [0.19 +/- 0.02 (SD) Lo/S] were produced by maximizing the mean power output (Wmax = 210 +/- 27 mW/cm2) that could be developed over 1.5 s when displacement was approximately 0.30 Lo. Initial peak isometric tension was 1.98 kg/cm2, whereas initial peak isovelocity tension was 1.84 kg/mc2 (P less than 0.01) or 93% of initial isometric tension. Fatigue trials of 5 min were conducted on muscles contracting at a constant duty cycle (0.43). At the end of the trials, peak isovelocity tension had fallen to 50% of initial isometric tension (P less than 0.01), whereas peak isometric tension had only fallen by 27%. These results indicate that muscle shortening during force production has a significant influence on diaphragm muscle fatigue. We conclude that the effects of shortening on fatigue must be considered in models of respiratory muscle function, because these muscles typically shorten during breathing.     

Assessment of cell-mediated hypersensitivity against coxsackievirus B3 viral-induced myocarditis utilizing hypertonic salt extracts of cardiac tissue.

Hypertonic KCl extracts prepared from heart tissues of adolescent CD-1 mice inoculated with coxsackievirus B3 (CVB3) were tested for antigenicity in evaluating cell-mediated sensitivity to CVB3 virus utilizing the agarose droplet cell-migration-inhibition assay. Immune mouse peritoneal exudate cells (IMPEC) from mice immunized against CVB3 virus and Freund's complete adjuvant were specifically inhibited in the cell-migration-inhibition assay with graded doses of KCl-extracted antigen and purified protein derivative (PPD). Unimmunized for "normal" mouse peritoneal exudate cells (NMPEC) were not inhibited in the presence of the CVB3 KCl extracts. KCl heart extracts from mice inoculated with a cardiotropic strain of antigenically distinct mengovirus failed to inhibit CVB3 IMPEC, and noncardiac KCl extracts of liver and spleen from CVB3-inoculated mice also failed to inhibit cellular migration of CVB3 IMPEC. Reciprocal specificity experiments utilizing KCl-extracted antigens from mice infected with antigenically distinct cardiotropic mengovirus failed to inhibit cellular migration of IMPEC from mice immunized against the mengovirus. Serum-blocking power experiments indicate the antigenic KCl extracts failed to bind virus-neutralizing antibodies, indicating absence of detectable quantities of virion antigens. The results indicate that inoculation of mice with CVB3 virus results in the appearance of a new antigen(s) in cardiac tissue reacting with CVB3-IMPEC, but not with mengovirus IMPEC.     

QiHong prevents death in coxsackievirus B3 induced murine myocarditis through inhibition of virus attachment and penetration

Viral myocarditis affects about 5% to 20% of the population. So far, there are not many effective antiviral treatments available. QiHong, the combination of the extracts from Astragali (Huangqi), Rhadiola rosea (Hongjingtian), and Sophora flavescens (Kushen), was developed based on laboratory research. The aim of this study was to investigate the effect and mechanism of QiHong on coxsackievirus B3 (CVB3)-induced myocarditis. The antiviral activity of QiHong in vitro was evaluated on HeLa and Vero cells infected by CVB3. Ribavirin was chosen as positive control. Our results showed that QiHong possessed potent antiviral effects on CVB3 by sodium 3'-[1-(phenylamino-carbonyl)-3, 4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid and plaque-forming assay (50% inhibitory concentrations [IC50] were 7.16 +/- 0.8 microg/ml and 2.63 +/- 0.5 microg/ml, respectively). The 50% cytotoxicity concentration (CC50) was 16-fold higher in QiHong-treated cells than in ribavirin-treated cells. Time course studies demonstrated that the antiviral effect of QiHong was mainly found during 0-4 hrs of infection, and it blocked the attachment and penetration of CVB3 into cells. In vivo 4-week-old male Balb/C mice were used and inoculated intraperitoneally with CVB3 suspension or normal saline. At 48 hrs after inoculation, the infected mice were gavaged with QiHong or ribavirin. On Day 6, myocardial virus titers were significantly lower in the QiHong-treated group than in the viral-infected groups. On Day 14, QiHong significantly ameliorated CVB3-induced myocardium necrosis; on Day 28, QiHong treatment increased survival rate 4-fold compared with CVB3-infected controls (64% vs. 16%; P < 0.05). The results showed that QiHong is a very promising potent antiviral agent with a highly significant favorable effect on survival and pathologic changes in CVB3-induced myocarditis with less toxicity than ribavirin. The antiviral activity of QiHong is at least partially due to an inhibitory effect on virus attachment and penetration.     

The pathogenesis of infection with minute virus of mice depends on expression of the small nonstructural protein NS2 and on the genotype of the allotropic determinants VP1 and VP2.

Neonatal C3H/He mice were oronasally inoculated with similar doses of four genotypes of minute virus of mice (MVM). MVMp, a fibroblast-specific variant, caused an asymptomatic infection. MVM(1035), a chimera which had the allotropic determinant of virulent MVMi inserted onto an MVMp background, caused a lethal infection and renal papillary infarcts, the hallmark of MVMi infection. MVMi(NS2-1990), the virulent lymphocyte-specific variant mutated to eliminate NS2 synthesis, was infectious but caused an asymptomatic infection. Sequential virus titration, histology, in situ hybridization with a full-length MVMi genomic probe, and immunohistochemistry for viral capsid antigen were used to compare the pathogenesis of infection with the four MVM genotypes. Infectious virus was recovered from multiple organs of mice infected with MVMi, MVMp, and MVM(1035) but not from mice infected with MVMi(NS2-1990). MVMp titers were lower than MVMi titers in all organs except the intestine. MVM(1035) titers were higher than MVMi titers in all organs except the blood. MVMp was localized to connective tissue elements of the intestine, to cells in mesenteric lymph nodes, and rarely to cells in other organs. MVM(1035) was localized to multiple organs and shared the same target cells, endothelium, lymphoid cells, and hematopoietic cells, as MVMi. MVM(1035) also replicated in external germinal cells of the cerebellum and smooth muscle cells of the stomach and colon, which were not targets of MVMi or MVMp infection. MVMi(NS2-1990) replicated to a limited degree in some MVMi target organs.     

Effect of low chloride on relaxation in hamster diaphragm muscle

With muscle fatigue the chloride (Cl-) conductance of the sarcolemmal membrane decreases. The role of lowered Cl- conductance in the prolongation of relaxation seen with fatigue was studied in isolated hamster diaphragm strips. The muscles were studied in either a Krebs solution or a low Cl- solution in which half of the NaCl was replaced by Na-gluconate. Short tetanic contractions were produced by a 160-ms train of 0.2-ms pulses at 60 Hz from which tension (T) and the time constant of relaxation were measured. Resting membrane potential (Em) was measured using KCl-filled microelectrodes with resistances of 15-20 M omega. Mild fatigue (20% fall in tension) was induced by 24-25 tetanic contractions at the rate of 2/s. There was no difference in Em or T in the two solutions, either initially or with fatigue. The time constant of relaxation was greater in low Cl- solution, both initially (22 +/- 3 vs. 18 +/- 5 ms, mean +/- SD, P less than 0.05) and with fatigue (51 +/- 18 vs. 26 +/- 7 ms, P less than 0.005). Lowering of sarcolemmal membrane Cl- conductance appears to play a role in the slowing of relaxation of hamster diaphragm muscle seen with fatigue.     

Direct interactions of coxsackievirus B3 with immune cells in the splenic compartment of mice susceptible or resistant to myocarditis.

In vitro replication of coxsackievirus B3 (CVB3) in cells of the immune system derived from uninfected adolescent A/J and C57BL/6J mice and replication of CVB3 in and association with immune cells from spleens of infected animals in vivo were assessed. Nonstimulated or mitogen-stimulated spleen cells were minimally permissive for viral replication during an 8-h period. Three days postinfection (p.i.), CVB3 RNA was localized in vivo to B cells and follicular dendritic cells of germinal centers in both A/J and C57BL/6J mice; however, extrafollicular localization was greater in C57BL/6J mice (P = 0.0054). Although the pattern of CVB3 RNA localization was different, the total load of infections virus (PFU per milligram of tissue) was not different. Splenic CVB3 titers (PFU per milligram of tissue) in both strains were maximal at day 3 or 4 p.i. and were back to baseline by day 7 p.i., with most infectious virus being non-cell associated. CVB3 titers (PFU per milligram of tissue) correlated directly with in situ hybridization positivity in splenic follicles and extrafollicular regions in both murine strains; however, follicular hybridization intensity was greater in A/J mice at day 5 p.i. (P = 0.021). Flow cytometric analysis demonstrated that 50.4% of total spleen cells positive for CVB3 antigen were B cells and 69.6% of positive splenic lymphocytes were B cells. Myocardial virus load in C57BL/6J mice was significantly lower than that in A/J mice at days 4 and 5 p.i. These data indicate that CVB3 replicates in murine splenocytes in vitro and in B cells and extrafollicular cells in vivo.     

Neutralizing Activity Induced by the Attenuated Coxsackievirus B3 Sabin3-like Strain Against CVB3 Infection

Coxsackievirus B3 (CVB3) causes viral myocarditis, and can ultimately result in dilated cardiomyopathy. There is no vaccine available for clinical use. In the present work, we assessed whether the Sabin3-like mutant of CVB3 could induce a protective immunity against virulent CVB3 Nancy and CVB4 E2 strains in mice by both oral and intraperitoneal (IP) routes. Serum samples, taken from mice inoculated with Sabin3-like, were assayed in vitro for their anti-CVB3 neutralizing activity. CVB3 Sabin3-like was highly attenuated in vivo and was able to induce an anti-CVB3 activity of the serum. However, at 4 days post-CVB3 challenge, significant increased titers of CVB3 neutralizing antibodies were detectable in the sera of immunized mice over the next 6 days. Non-immunized mice challenged with CVB3 Nancy had no anti-CVB3 activity in their sera until 10 days post-infection. CVB3 Nancy induced higher viral titers than did the mutant strain. There was no variation of the neutralizing activity of serum taken from mice immunized with CVB3 Sabin3-like and challenged with CVB4 E2, compared to non-immunized mice. Despite the fact that CVB3 and CVB4 are closely related viruses, virus-neutralizing activity clearly distinguish between these viruses. A variable and limited amount of pancreatic inflammation was seen in some mice 10 days after Sabin3-like inoculation by IP route, whereas there was no evidence of pancreatic damage in mice inoculated by oral route. All immunized mice were protected from myocarditis and pancreatitis at 8 days post-challenge with CVB3 or CVB4 E2. These findings strongly suggest that the mutant strain could be considered a candidate for an attenuated CVB3 vaccine.     

Coxsackievirus-B3-induced myocarditis: virus receptor antibodies modulate myocarditis

Two variants of coxsackievirus group B, type 3 (CVB3) differ in ability to induce myocarditis in Balb/cCUM mice. Infection with the highly pathogenic variant (CVB3M) stimulates autoimmunity to normal cardiocyte antigens, and tissue injury results primarily from an autoreactive cytolytic T lymphocyte (ACTL). Animals infected with the less pathogenic CVB3o variant do not develop ACTL, although CVB3o replicates to high titers in the heart and polyclonal neutralizing antisera fail to distinguish between the two variant virions. The present study uses two IgM mAb derived by fusing spleen cells from CVB3M-infected mice with NS-1 cells. These mAb investigate important differences between the virus variants that may explain why only selected infections trigger autoimmunity. mAb 8A6 is a virus-neutralizing antibody that prevents infection of HeLa cells and cultured cardiocytes by attaching to the virus. mAb 10A1 also interferes with infection but presumably reacts to the virus receptor on the susceptible cells and shows little or no binding to the virions. While 8A6 is equally effective in neutralizing both CVB3o and CVB3M, suggesting that antigenic epitopes on both variants are either identical or highly cross-reactive, 10A1 distinguishes between the variants, suggesting that the pathogenic and less pathogenic viruses use distinct cell surface receptors. Competitive binding studies using radiolabeled CVB3M and either of the unlabeled variants confirm this hypothesis. Both mAb effectively prevent CVB3M-induced cardiac damage in vivo. mAb 10A1 also inhibits autoreactive ACTL lysis of cardiocytes, indicating that the autoimmune effectors may recognize the virus receptor, and that the receptor utilized by a virus may prove important in triggering auto-sensitization.     

5'-Terminal deletions occur in coxsackievirus B3 during replication in murine hearts and cardiac myocyte cultures and correlate with encapsidation of negative-strand viral RNA

Adult human enteroviral heart disease is often associated with the detection of enteroviral RNA in cardiac muscle tissue in the absence of infectious virus. Passage of coxsackievirus B3 (CVB3) in adult murine cardiomyocytes produced CVB3 that was noncytolytic in HeLa cells. Detectable but noncytopathic CVB3 was also isolated from hearts of mice inoculated with CVB3. Sequence analysis revealed five classes of CVB3 genomes with 5' termini containing 7, 12, 17, 30, and 49 nucleotide deletions. Structural changes (assayed by chemical modification) in cloned, terminally deleted 5'-nontranslated regions were confined to the cloverleaf domain and localized within the region of the deletion, leaving key functional elements of the RNA intact. Transfection of CVB3 cDNA clones with the 5'-terminal deletions into HeLa cells generated noncytolytic virus (CVB3/TD) which was neutralized by anti-CVB3 serum. Encapsidated negative-strand viral RNA was detected using CsCl-purified CVB3/TD virions, although no negative-strand virion RNA was detected in similarly treated parental CVB3 virions. The viral protein VPg was detected on CVB3/TD virion RNA molecules which terminate in 5' CG or 5' AG. Detection of viral RNA in mouse hearts from 1 week to over 5 months postinoculation with CVB3/TD demonstrated that CVB3/TD virus strains replicate and persist in vivo. These studies describe a naturally occurring genomic alteration to an enteroviral genome associated with long-term viral persistence.