Effects of nutritional antioxidants and other dietary constituents on coxsackievirus-induced myocarditis.
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Biomedical subjects
M A Beck
Publications and source records attributed to M A Beck.
Interacting nutritional and infectious etiologies of Keshan disease. Insights from coxsackie virus B-induced myocarditis in mice deficient in selenium or vitamin E.
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, 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).
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Increased virulence of coxsackievirus B3 in mice due to vitamin E or selenium deficiency.
Nutrition has long been known to affect the ability of the host to respond to infectious disease. Widespread famines are often accompanied by increased morbidity and mortality due to infectious diseases. The currently accepted view of the relationship between nutrition of the host and its susceptibility to infectious disease is one of a direct relationship with host immune status. That is, if the nutritional status of the host is poor-due to either single or multiple nutrient deficiencies-then the functioning of the host immune system is compromised. This impairment of the immune response will lead to an increased susceptibility to infectious disease. Clearly, the immune response has been shown to be weakened by inadequate nutrition in many model systems and in human studies. However, what about the effect of host nutrition on the pathogen itself? Our laboratory has shown, using a mouse model of coxsackievirus-induced myocarditis, that a host deficiency in either selenium or vitamin E leads to a change in viral phenotype, such that an avirulent strain of the virus becomes virulent and a virulent strain becomes more virulent. The change in phenotype was shown to be due to point mutations in the viral genome. Once the mutations occur, the phenotype change is stable and can now be expressed even in mice of normal nutriture. Our results suggest that nutrition can affect not only the host, but the pathogen as well, and demonstrate a new model of relating host nutritional effects to viral pathogenesis.
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Rapid genomic evolution of a non-virulent coxsackievirus B3 in selenium-deficient mice.
Keshan disease, an endemic cardiomyopathy in China, can be prevented with selenium (Se) supplementation. However, the seasonal and annual nature of the disease suggests that an infectious co-factor is required along with a deficiency in Se. Using a murine model of coxsackievirus B3 (CVB3)-induced myocarditis, Se-deficient mice were shown to be more susceptible to the cardiopathologic effects of the virus. In addition, a normal benign strain of CVB3 becomes virulent in Se-deficient mice. This change in virulence was shown to be due to point mutations in the viral genome. Although the mechanism of the viral mutation is not known, the oxidative stress status of the Se-deficient host may play a role, either by directly affecting the virus and/or affecting host immune defenses.
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Requirement of MIP-1 alpha for an inflammatory response to viral infection.
Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is a chemokine that has pro-inflammatory and stem cell inhibitory activities in vitro. Its biologic role in vivo was examined in mice in which the gene encoding MIP-1 alpha had been disrupted. Homozygous MIP-1 alpha mutant (-/-) mice were resistant to Coxsackievirus-induced myocarditis seen in infected wild-type (+/+) mice. Influenza virus-infected -/- mice had reduced pneumonitis and delayed clearance of the virus compared with infected +/+ mice. The -/- mice had no overt hematopoietic abnormalities. These results demonstrate that MIP-1 alpha is an important mediator of virus-induced inflammation in vivo.
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Rapid genomic evolution of a non-virulent coxsackievirus B3 in selenium-deficient mice results in selection of identical virulent isolates.
Previous work from our laboratory demonstrated that selenium deficiency in the mouse allows a normally benign (amyocarditic) cloned and sequenced Coxackievirus to cause significant heart damage. Furthermore, Coxsackievirus recovered from the hearts of selenium-deficient mice inoculated into selenium-adequate mice still induced significant heart damage, suggesting that the amyocarditic Coxsackievirus had mutated to a virulent phenotype. Here we report that sequence analysis revealed six nucleotide changes between the virulent virus recovered from the selenium-deficient host and the avirulent input virus. These nucleotide changes are consistent with known differences in base composition between virulent and avirulent strains of Coxsackievirus. To the best of our knowledge, this is the first report of a specific nutritional deficiency driving changes in a viral genome, permitting an avirulent virus to acquire virulence due to genetic mutation.
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Vitamin E and selenium: contrasting and interacting nutritional determinants of host resistance to parasitic and viral infections.
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Benign human enterovirus becomes virulent in selenium-deficient mice.
Coxsackieviruses have been implicated as possible co-factors in the etiology of the selenium (Se)-responsive cardiomyopathy known as Keshan disease. Here we report that a cloned and sequenced amyocarditic coxsackievirus B3 (CVB3/0), which causes no pathology in the hearts of Se-adequate mice, induces extensive cardiac pathology in Se-deficient mice. CVB3/0 recovered from the hearts of Se-deficient mice inoculated into Se-adequate mice induced significant heart damage, suggesting mutation of the virus to a virulent genotype. We demonstrate the important role of host nutritional status in determining the severity of a viral infection.
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Increased virulence of a human enterovirus (coxsackievirus B3) in selenium-deficient mice.
Coxsackievirus B3 (CVB3/20)-induced myocarditic lesions occurred more quickly and were more severe and virus titers in heart and liver were higher in selenium (Se)-deficient than Se-adequate mice. NK cell activity and serum neutralizing antibody titers were similar in both Se-adequate and -deficient CVB3/20-infected mice; however, lymphocyte proliferation to both mitogen and antigen was decreased in Se-deficient mice. CVB3/20 isolated from Se-deficient donor mice and inoculated into Se-adequate recipient mice induced severe myocarditis. In contrast, CVB3/20 isolated from Se-adequate donor mice and inoculated into Se-adequate recipient mice induced only moderate myocarditis, similar to that caused by the original virus stock. Thus, the general population of CVB3/20 virions, as a consequence of replicating in an Se-deficient host, underwent a phenotypic change to increased virulence. These results have important implications for the emergence of virulent viruses.
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Vitamin E deficiency intensifies the myocardial injury of coxsackievirus B3 infection of mice.
Feeding a vitamin E-deficient diet increases pathology in hearts of mice infected with a myocarditic coxsackievirus B3 (CVB3/20). Hearts from infected mice fed a vitamin E-deficient diet rich in highly unsaturated fat (menhaden oil) exhibited more severe pathology than hearts from infected mice fed a vitamin E-deficient diet based largely on saturated fat (lard). Furthermore, a cloned and sequenced amyocarditic coxsackievirus B3 (CVB3/0), which caused little or no pathology in the hearts of vitamin E-supplemented mice, induced extensive cardiac pathology in vitamin E-deficient mice. In infected mice, both mitogen and antigen responses were depressed by vitamin E deficiency, although neutralizing antibody responses were unaffected. Natural killer cell responses were comparable in infected mice fed a lard-based diet with or without supplemented vitamin E. However, a menhaden oil-based diet, whether supplemented with vitamin E or not, significantly depressed natural killer cell activity in infected mice compared with mice fed the lard-based diet. Coxsackievirus B3/0 recovered from the heart of a vitamin E-deficient donor mouse, passaged one time onto HeLa cells, caused significant heart damage when passed back into vitamin E-supplemented recipient mice, demonstrating that the amyocarditic CVB3/0 had changed to a virulent phenotype. Enhanced virulence was also seen with CVB3/20 virus similarly passaged in a vitamin E-deficient donor. Our work demonstrates the important role of host nutritional antioxidant status in determining the severity of certain viral infections.
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Comoviruses and enteroviruses share a T cell epitope.
An in vitro murine T cell proliferation assay was used to determine whether an antigenic epitope(s) recognized by enterovirus-immune T cells is held in common between plant comoviruses and human enteroviruses. Splenocytes isolated from C3H/HeJ mice infected with coxsackievirus B3 (CVB3) proliferated in vitro not only against a variety of enterovirus (CVB2, CVB3, CVB6, CVA16, PV1) antigens, but against comovirus (CPMV, BPMV) antigens as well. Splenocytes from mice inoculated with bean pod mottle virus (BPMV) also proliferated in response to comoviral and enteroviral antigens in vitro. However, if the viral inocula were highly purified prior to inoculation, then the splenocyte response was generated only against the group used to inoculate, suggesting that the epitope shared between the comoviruses and the enteroviruses resided in the nonstructural region. B (nonstructural) and M (structural) genomic segments of CPMV were translated in rabbit reticulocyte lysates and used as in vitro antigens. Splenocytes from mice inoculated with live CVB3 proliferated in response to the B-RNA-encoded but not the M-RNA-encoded polypeptides, confirming the nonstructural coding region location of the common epitope. Comparison of predicted amino acid sequences in the nonstructural coding regions of the comoviruses and picornaviruses suggested a potentially immunogenic linear epitope in protein 2C. The consensus peptide LEEKGI was synthezized and shown to be immunogenic for both BPMV- and CVB3-immune splenocytes.
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Progress and challenges in the pathological diagnosis of myocarditis.
The pathological diagnosis of myocarditis rests on well-described histopathological criteria. Appreciation for the disease-specific sensitivity of the endomyocardial biopsy, as well as the phenotypical and functional nature of inflammatory infiltrates, will enhance the clinical utility of the biopsy technique. New information regarding the role of enteroviruses in immunological sensitization and the study of enteroviruses in murine models and human patients are fostering new perspectives and routes of investigation of appropriate therapeutic interventions for myocarditis.
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A molecular and serologic evaluation of enteroviral involvement in human myocarditis.
Murine coxsackie B virus infection models of myocarditis and numerous human serologic studies associating elevated enterovirus-specific IgM titers with the clinical diagnosis of myocarditis have been used to support an etiologic role for enteroviruses in human myocarditis. Of the human enteroviruses, coxsackie B viruses (CVB) are the enterovirus group most commonly associated with the human disease. While hybridization probes exist to detect most, if not all, human enteroviruses, no probe capable of specifically detecting an enteroviral group (such as the CVB) to the exclusion of all others has been described to date. Thus, to test the hypothesis that enteroviral involvement in human myocarditis is commonplace, we examined a case series of human myocarditic heart tissues for enteroviruses by in situ hybridization using a generic enterovirus probe. These results were then compared with CVB-specific IgM levels in the cognate patient sera. Comparison of the hybridization data with the CVB-specific IgM levels in the cognate sera yielded no valid correlation between the detection of enteroviral RNA and specificity of CVB identification in any patient. The data are consistent with common enterovirus infections in humans and a possible etiologic role in myocarditis but do not support a specific etiologic role for CVB in this study group.
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Evidence for a group-specific enteroviral antigen(s) recognized by human T cells.
Human peripheral blood mononuclear cells from 15 normal, healthy adult volunteers proliferated in vitro against a panel of enteroviral antigens, including coxsackievirus B3, coxsackievirus B2, coxsackievirus B6, coxsackievirus A16, and poliovirus 1. No proliferation against the cardiovirus encephalomyocarditis virus occurred. Lymphocytes obtained from cord blood drawn from seven neonates were uniformly nonresponsive to enteroviral antigens. Although serum neutralization antibody titers indicated different exposure histories of the volunteers, only one had a titer against coxsackievirus B6, a rare isolate in the United States. The peripheral blood mononuclear cells from each volunteer responded in vitro to each enterovirus tested even though not all individuals had serum neutralizing antibody against each virus. The predominant cell type responding in vitro was the CD4+ T cell. Denaturation of viral antigen by Formalin did not prevent the recognition of the common group antigen by the T cells, indicating that noninfectious virus can also serve as antigen. These data demonstrate that human T cells recognize a common enterovirus group antigen(s).
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Echovirus 22 is an atypical enterovirus.
Although echovirus 22 (EV22) is classified as an enterovirus in the family Picornaviridae, it is atypical of the enterovirus paradigm, typified by the polioviruses and the coxsackie B viruses. cDNA reverse transcribed from coxsackievirus B3 (CVB3) RNA does not hybridize to genomic RNA of EV22, and conversely, cDNA made to EV22 does not hybridize to CVB3 genomic RNA or to molecular clones of CVB3 or poliovirus type 1. EV22 cDNA does not hybridize to viral RNA of encephalomyocarditis virus or to a molecular clone of Theiler's murine encephalomyelitis virus, members of the cardiovirus genus. The genomic RNA of EV22 cannot be detected by the polymerase chain reaction using generic enteroviral primers. EV22 does not shut off host cell protein synthesis, and the RNA of EV22 is efficiently translated in vitro in rabbit reticulocyte lysates. Murine enterovirus-immune T cells recognize and proliferate against EV22 as an antigen in vitro, demonstrating that EV22 shares an epitope(s) common to enteroviruses but not found among other picornaviruses.
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Secondary enterovirus infection in the murine model of myocarditis. Pathologic and immunologic aspects.
Enteroviruses are implicated as etiologic agents in the inflammatory diseases myocarditis and polymyositis. In this report, we show that a previous enterovirus exposure in mice can influence development of myocardial inflammation with a second enteroviral exposure. Inoculation of 25-day-old male C3H/HeJ mice with 10(3) or 10(5) plaque-forming units (PFU) of infectious or ultra violet (UV)-inactivated coxsackievirus B2 (CVB2), followed by inoculation 28 days later with 10(5) PFU of a myocarditic variant of coxsackievirus B3 (CVB3-m) results in more intense myocardial inflammation and injury than is seen in age-matched mice inoculated with CVB3-m alone. More severe disease occurs with the lower primary dose of CVB2. Neutralizing antibody to CVB2 is detected early after primary inoculation and neutralizing antibody to CVB3 is first detected 5 days after secondary inoculation. In vitro proliferation of splenocytes from mice inoculated with one or both viruses occurs in response to both CVB2 and CVB3 antigens. We recently demonstrated that murine T cells are capable of recognizing an enterovirus group antigen. Thus cell-mediated immune responses to a conserved antigenic epitope(s) among the enteroviruses may be involved in the exacerbation of myocardial inflammatory disease during a second enterovirus infection. The secondary infection model described here may more accurately mirror virus-induced myocarditis in the human population because the majority of adults have been exposed to several enteroviruses before induction of disease.
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Regulation of lymphokine response during reinfection by influenza virus. Production of a factor that inhibits lymphokine activity.
Mononuclear cells, obtained from the spleens and lungs of influenza virus-seropositive C57BL/6 mice at 2 to 4 days after re-infection with homologous virus (strain A/Bangkok/1/79), produced a low m.w. factor in vitro that prevents the biologic expression, but not production, of the lymphokine, leukocyte migration inhibition factor (LIF). The low m.w. factor inhibited LIF activity without destroying the LIF molecule inasmuch as simple dialysis restored lymphokine activity to culture supernatants. Production of the low m.w. factor was observed from 2 to 4 days after re-infection, at which time the delayed-type hypersensitivity response to viral Ag was suppressed. In contrast, LIF was produced by splenocytes and lung mononuclear cells obtained at all times tested after re-infection (from 2 to 30 days). Production of the low m.w. factor required re-infection of influenza A virus-seropositive mice with type A virus; re-infection with influenza B virus failed to induce production. Ag specificity was also required in vitro for splenocytes to produce the factor; cells from type A virus-re-infected mice required type A Ag stimulation. Cell depletion studies with mAb plus C revealed that macrophages and T cells along with Ag stimulation were required for factor production by spleen cells. However, mononuclear cells obtained within 4 days from the lungs of re-infected mice did not require in vitro Ag stimulation for production of the low m.w. factor, and factor production was dependent upon the presence of CD4+ (L3T4) cells in the culture. Fractionation of culture supernatants over a Sephadex G-50 column indicated that the factor had a molecular mass of 2 to 3 kDa, and by FPLC chromatofocusing over a Mono P column, the factor eluted at a pH of approximately 8.2. Thus, re-exposure of influenza virus-seropositive mice to homologous virus resulted in the production of a low m.w. factor that prevented the biologic expression of LIF, but not its production. Lymphokines are an important component of the delayed-type hypersensitivity response; the presence of mononuclear cells secreting a low m.w. factor and LIF concomitantly at the site of virus replication (lungs) and the capacity of the factor to block the biologic expression of LIF in vitro suggest that the factor may have a role in the regulation of a delayed-type hypersensitivity response in vivo during re-infection.
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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.
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