Coxsackievirus Group Research Articles

Susceptibility of RD-9H8 cells derived from RD cells to enteroviruses and its application for measuring neutralizing antibodies against coxsackievirus group B

We carried out cloning of RD cells and obtained cloned cell line RD-9H8, which is highly sensitive to enteroviruses. Cytopathic effect (CPE) of RD-9H8 cells was more remarkable than that of original RD cells. The RD-9H8 cells were used to measure the neutralizing antibodies to coxsackievirus group B in sera from 93 medical students collected in Oct. 1990 at Iwate Medical University. The proportions of confirmed positive sera were coxsackievirus type B1 44.1%, type B2 50.5%, type B3 46.2%, type B4 64.5%, type B5 53.8% and type B6 4.4%.

An attenuated variant of Coxsackievirus B3 preferentially induces immunoregulatory T cells in vivo.

BALB/c mice infected with the Woodruff variant of coxsackievirus group B type 3 (CVB3W) develop myocarditis mediated by autoimmune cytolytic T lymphocytes. A variant of CVB3W (designated H3-10A1) which infects the myocardium but induces minimal mortality of myocarditis compared to the parental virus was selected. Although H3-10A1 infections stimulate normal CTL responses to CVB3-infected myocytes, the autoimmune response to myocardial antigens is absent. Treatment of H3-10A1-infected mice with 50 mg of cyclophosphamide per kg of body weight, a treatment which preferentially eliminates suppressor cells, allows both the development of the autoimmune cytotoxic T-lymphocyte response and the expression of myocarditis. Similar treatment of CVB3W-infected mice had no effect on the disease. The presence of the immunoregulatory cells was confirmed by adoptive transfer of T lymphocytes from either H3-10A1 or CVB3W-infected donor mice into syngeneic CVB3W-infected recipients. Animals given H3-10A1-immune cells had minimal myocardial inflammation, while animals given CVB3W-immune lymphocytes developed enhanced cardiac disease. Elimination of the T-lymphocyte population from the donor cells prior to transfer abrogated suppression with the H3-10A1-immune population, showing that immunoregulation depended upon T lymphocytes. Both H3-10A1 and CVB3W have cross-reactive epitopes between the adenine translocator protein and the virion which are indicative of antigenic mimicry and may be the basis for the autoimmunity to cardiac antigens. These results suggest that immunoregulatory T cells may be primarily responsible for the nonpathogenicity of the H3-10A1 variant.

Functional diversity in vascular endothelial cells: role in coxsackievirus tropism

Six plaque-purified virus isolates were obtained from liver and heart tissues of a DBA/2 mouse infected 7 days earlier with 10(4) PFU of coxsackievirus group B type 3. Each virus isolate was assayed in vitro for infectivity to vascular endothelial cells (VEC) of the liver, lungs, and heart. Both the percentage of VEC infected and the mean progeny PFU produced per infected VEC were determined. Virus isolates from the heart showed greater infectivity and replication in heart VEC than in VEC derived from either the liver or lungs. Similarly, virus isolated from the liver preferentially infected liver VEC. Virus receptor expression varied between VEC populations, as demonstrated by binding studies with a [35S]methionine-radiolabeled heart virus and by enzyme-linked immunoadsorption assay studies with a monoclonal antibody to the coxsackievirus group B type 3 receptor on heart tissue. Finally, the heart and liver virus isolates were injected (10(4) PFU) intraperitoneally into BALB/c mice. After 7 days, the animals were sacrificed, and the hearts, livers, and lungs were evaluated for tissue injury and virus concentrations. Viruses originally isolated from the heart preferentially infected the heart when reinjected into animals and caused severe myocarditis. Viruses originally derived from the liver most consistently reinfected the liver, although significant virus concentrations were also detected in the heart. The liver virus isolates, however, were incapable of causing myocarditis. Thus, selective tropism of viruses for particular organs in vivo corresponds to the ability of these isolates to infect VEC in vitro.

Molecular detection and identification of enteroviruses using enzymatic amplification and nucleic acid hybridization

Analysis of enteroviral genomes has revealed that the 5' nontranslated region is highly conserved, providing consensus sequences for the design of oligonucleotides which should anneal to most, if not all, human enteroviral RNAs. We designed and used a pair of such generic primers to enzymatically amplify cDNA from coxsackievirus group B types 1 through 6, poliovirus types 1 through 3, 4 coxsackievirus A types, and 29 echoviruses. The polymerase chain reaction (PCR) products generated with these enteroviral primers were analyzed by agarose gel electrophoresis, Southern blotting, or slot blot hybridization. A genotype-specific PCR was used to detect coxsackievirus B3, to the exclusion of other enteroviruses, by using a coxsackievirus B3 genome-specific primer pair that was derived from sequences coding for part of a capsid protein. A technique is demonstrated by which individual genotypes, for which no sequence information is known, can be identified by high-criterion hybridization analysis following amplification with generic enterovirus PCR primers.

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.

Outbreak of herpangina associated with coxsackievirus B3 infection

An outbreak of acute febrile illness was observed in summer, 1987, in a welfare home in which 31 healthy infants were accommodated. Within a 5-day period 25 infants (81%) acquired a febrile illness. Coxsackievirus B3 was isolated from 16 (64%) of 25 throat swabs. In the patients in whom viral culture was negative or not performed, 6 were serologically identified as having a coxsackievirus B3 infection. Among 22 patients identified as having a coxsackievirus B3 infection 7 had typical herpangina and the others had pharyngitis with or without a few small vesicles. Serum alpha-interferon was detected in all but 2 cases (one with proved infection and another with indefinite infection). Herpangina can be associated with coxsackievirus B3 as well as with the more frequently associated coxsackievirus Group A; this explosive type of outbreak might be transmitted by a small particle aerosol.

Clinical and experimental aspects of viral myocarditis.

Picornaviruses are frequently implicated as the etiological agents of acute myocarditis. This association is based historically on serological evidence of rising antibody titers to specific pathogens and more recently on identification of viral genomic material in endocardial biopsy specimens through in situ hybridization. Only rarely is infectious virus isolated from either the patient or the heart during periods of maximum myocardial inflammation and injury. Thus, despite a probable viral etiology, much interest centers on the role of the immune system in cardiac damage and the likelihood that the infection triggers an autoimmune response to heart-specific antigens. Heart-reactive antibodies and T cells are found in most myocarditis patients, and immunosuppressive therapy has proven beneficial in many, though not all, cases. Furthermore, murine models of coxsackievirus group B type 3-induced myocarditis also demonstrate that virus infection initiates autoimmunity and that these autoimmune effectors are predominately responsible for tissue injury. How virus-host interactions overcome presumed self-tolerance to heart antigens is discussed, and evidence supporting various theories of virus-initiated autoimmunity and disease pathogenesis are delineated.

Chronic neonatal coxsackie myocarditis.

A baby girl, with a birth weight of 2540 g, developed myocarditis with gross, permanent myocardial calcification. The clinical course was progressively downhill, and she died at 14 months of age. Neutralisation antibody to Coxsackie virus group B1 rose from 1:80 after delivery to 1:1280 at age 5 months. The protracted course of this infant's disease represents a new clinical form of fatal neonatal Coxsackie virus group B infection.

下水におけるコクサッキーB群ウイルスの疫学的観察

Different types of coxsackieviruses group B were isolated from the sewage consecutively annually from January 1982 to March 1987: type 3 in 1982, type 4 in 1983, type 2, 4 and 5 in 1984, type 3 in 1985, and type 4 in 1986 were found. These viruses caused large epidemics among children aged 1 to 4 and proximities. Herald waves for the epidemics were observed six times during this period, and the viruses were detected in a brief period of isolation. With respect to the periodicity of these viruses, the cycles for types 3 and 4 were 2 to 3 years, and longer cycles seemed to be exhibited by types 1, 2, and 5. The onset and termination of the epidemics will only be grasped by surveys throughout the year. HEp-2 cells exhibited the best sensitivity to the viruses.

Clinical and experimental aspects of viral myocarditis.

Picornaviruses are frequently implicated as the etiological agents of acute myocarditis. This association is based historically on serological evidence of rising antibody titers to specific pathogens and more recently on identification of viral genomic material in endocardial biopsy specimens through in situ hybridization. Only rarely is infectious virus isolated from either the patient or the heart during periods of maximum myocardial inflammation and injury. Thus, despite a probable viral etiology, much interest centers on the role of the immune system in cardiac damage and the likelihood that the infection triggers an autoimmune response to heart-specific antigens. Heart-reactive antibodies and T cells are found in most myocarditis patients, and immunosuppressive therapy has proven beneficial in many, though not all, cases. Furthermore, murine models of coxsackievirus group B type 3-induced myocarditis also demonstrate that virus infection initiates autoimmunity and that these autoimmune effectors are predominately responsible for tissue injury. How virus-host interactions overcome presumed self-tolerance to heart antigens is discussed, and evidence supporting various theories of virus-initiated autoimmunity and disease pathogenesis are delineated.

Acute Myocarditis

In a prospective study, 57 patients with a preliminary diagnosis of myocarditis were investigated. Twenty-four patients were considered to have an acute myocarditis, 14 had a suspected myocarditis, while in 19 patients myocarditis was excluded. Episodes of frequent supraventricular and/or ventricular extrasystoles during hospital stay were seen in 8/24 cases (33%) with myocarditis and in 1/19 cases (5%) without myocarditis. On follow-up 1 month later, no supraventricular extrasystoles were observed in either group. Echocardiographic signs consistent with left ventricular insufficiency were noted in 7/24 cases (29%) with myocarditis, in 1/14 cases (7%) with suspected myocarditis and in no case without myocarditis. With a "routine" serologic test battery covering influenza viruses A and B, adenovirus, Coxsackie virus group B, ECHO viruses, Chlamydia psittaci, Mycoplasma pneumoniae and hemolytic streptococci group A, a possible etiology could be documented in 9/24 cases (38%) with myocarditis and in 4/19 cases (21%) without myocarditis. Enterovirus-specific IgM was detected with solid-phase reverse immunosorbent test (SPRIST) in 12/23 (48%) cases with myocarditis and in 3/16 cases (19%) without myocarditis. In SPRIST-IgM-positive cases, IgM antibodies were detected in 15/20 (75%) of the sera taken on admission. The overall serological results indicated a recent infection in 16/24 cases (67%) with myocarditis and in 5/19 cases (26%) without myocarditis (p less than 0.05).

Influence of sex hormones on coxsackie virus group B, type 3 induced myocarditis in Balb/c mice

Susceptibility to coxsackie virus group B, type 3 (CVB3) induced myocarditis changes with age. Very young (< 4 weeks) Balb/c mice show minimal cardiac injury after infection, while young adult males (6–12 weeks) develop severe myocarditis. Age-matched females remain largely protected. As the mice continue to age, susceptibility in females gradually increases while that in males decreases, ultimately resulting in equal myocardial damage between the sexes by 40 weeks of age. Enhanced myocarditis in old females correlates withincreased humoral and cellular immunity to the virus and heart antigens and results from decreased oestrogen levels in these animals.

Susceptibility of Aedes albopictus C6/36 cells to viral infection.

The susceptibility of the C6/36 clone of Aedes albopictus monolayer cell cultures was determined with 46 prototype viruses passed through three subcultures. Viral growth was confirmed by titration of the passage material in other susceptible host systems. Nineteen viruses demonstrated good growth in C6/36 cells: coxsackievirus group A type 10 and group B types 2, 3, 4, and 5; enterovirus 69; mumps virus; poliovirus types 1 to 3; reovirus types 1 to 3; vaccinia virus; dengue virus type 2; eastern equine encephalomyelitis virus; La Crosse virus; Rocio virus; and St. Louis encephalitis virus. Ten viruses did not adapt to growth in the C6/36 cultures. Seventeen other virus strains displayed only limited growth which was primarily restricted to the initial C6/36 passage or was detected by hemagglutinin reactions without observable cell degeneration. Of the 46 viruses, 33 (72%) were capable of initiating infection with a demonstrable cytopathic effect in the initial C6/36 passage. Hemagglutination or complement fixation titers or both were obtained with dengue virus type 2, eastern equine encephalomyelitis virus, La Crosse virus, mumps virus, reovirus types 1 to 3, and Rocio, St. Louis encephalitis, and vaccinia viruses.

Variation in susceptibility of Balb/c mice to coxsackievirus group B type 3-induced myocarditis with age

The severity of cardiac lesions in coxsackievirus group B, type 3 (CVB3)-infected Balb/c mice depends upon both the age and the sex of the animal at the time of viral inoculation. Suckling animals (1–3 weeks old) of either sex develop few cardiac lesions. Thereafter, males rapidly demonstrate increasing disease susceptibility peaking at 16–18 weeks old and then decreasing susceptibility from 20 to 40 weeks of age. Female susceptibility increases much more gradually and myocarditis in this sex never reaches maximal levels as seen in males. Increased susceptibility correlates with virus concentrations in the heart and anti-CVB3 titers in the serum. Cardiac injury is dependent on functional T lymphocytes since treatment of the animals with rabbit antimouse thymocyte serum abrogates inflammation and myocyte necrosis. Sex-associated steroid hormones influence both virus concentrations and immune responses in mice and are probably responsible for variations in disease susceptibility throughout the animal's life.

Coxsackievirus group B replication in cultured fetal baboon aortic smooth muscle cells.

All six coxsackievirus B (CVB) serotypes replicated to various extents in fetal baboon aortic smooth muscle cells (SMC) in culture. CVB3 and CVB4 replicated to the highest titers and induced no cytopathology at the level of light microscopy. Maximum yields of CVB3 were produced between 12 and 24 hr postinoculation. Up to 15% of SMC cells became infected, as determined by immunofluorescence assays with anti-CVB3 antiserum, yet overall cell division in infected cultures did not differ from infected SMC cultures. Electron microscopy of CVB3-inoculated SMC cultures revealed changes in some cells: viruslike particles, secondary lysosomes containing dense bodies, and peripheral nuclear chromatin condensation. CVB3 replicated well in SMC passages up to the eighth, but did not replicate in eleventh-passage cells. Because of the cardiotropic and myotropic potential of this virus and its ability to replicate in aortic SMC with associated ultrastructural alterations, CVB3 (and other CVB) should be further examined as an etiologic agent(s) that could induce atherosclerosis.

Demonstration of suppressor cells in coxsackievirus group B, type 3 infected female Balb/c mice which prevent myocarditis

Coxsackievirus group B type 3 (CVB3) induces myocarditis in male Balb/c mice but produces little cardiac injury in females. Males develop cytolytic T lymphocytes (CTL) reactive to heart antigens which primarily cause the inflammation and cardiac injury observed in the disease. Infected female mice lack this CTL response because they rapidly produce suppressor cells inhibiting both cellular immunity and cardiac inflammation. Four lines of evidence demonstrate suppressor cells in females. First, females develop myocarditis when treated with low-dose cyclophosphamide under conditions known to preferentially eliminate suppressor cells but not other immune cells. Second, lymphocytes obtained from females at various times after infection prevent myocarditis when adoptively transferred into CVB3-infected males. Virus concentrations in the hearts of males receiving immune female cells and control males were equivalent. Thus protection did not result from accelerated virus elimination in recipient males. Third, CTL from CVB3 infected male mice could induce myocarditis in infected T-lymphocyte depleted but not in intact females suggesting the presence of an inhibitory T cell in the intact animals. Finally, male lymphocytes cultured on heart cell monolayers for 5 days generate significant cytolytic activity to myocyte targets. CTL generation could be inhibited by co-culture of the male cells with immune female lymphocytes. Nonimmune female cells were not inhibitory.

Coxsackievirus Group B Antibodies in the Ventricular Fluid of Infants with Severe Anatomic Defects in the Central Nervous System

Ventricular fluids from four of 28 newborn infants who were initially seen with severe congenital anatomic defects in the central nervous system contained neutralizing antibody to at least one serotype of coxsackieviruses group B. Two of the four infants with anti-coxsackieviruses group B antibody in the ventricular fluid did not have a detectable level of the same antibody(ies) in their serum. The ventricular fluid of one of the infants had immunoglobulin M neutralizing antibody directed against coxsackievirus B6. Of 11 mother-infant pairs that had neutralizing antibody to coxsackieviruses group B in both sera, nearly half had antibodies directed against more than one serotype. These data suggest the possibility of an association between congenital infections with coxsackieviruses group B and rare severe CNS defects.

1095 COXSACKIEVIRUSES GROUP B ANTIBODIES IN THE VENTRICULAR CEREBROSPINAL FLUID OF INFANTS WITH SEVERE ANATOMICAL DEFECTS IN THE CENTRAL NERVOUS SYSTEM

Coxsackieviruses group B (CVB) are ubiquitous viruses which rarely may be involved in congenital diseases and are known to cause central nervous system infections. We set out to determine whether the CVB might be associated with severe congenital anatomical defects in the central nervous system (CNS), mainly congenital hydrocephalus, in infants. Ventricular cerebrospinal fluids from 4 of 28 newborn infants presenting with severe problems of the CNS contained neutralizing antibody to at least one serotype of the CVB. Two of the 4 infants with anti-CVB antibody in the ventricular fluid did not have a detectable level of the same antibody(ies) in their serum. Hydranencephaly was diagnosed in 2 of these same 4 infants. The ventricular fluid of one of the infants had IgM neutralizing antibody directed against coxsackievirus B6. Of 11 mother/infant pairs which had neutralizing antibody to CVB in both sera, almost half had antibodies directed against more than one serotype. Neutralizing antibodies to all 6 serotypes except coxsackievirus B5 were found. Isolation of a virus from the ventricular cerebrospinal fluids was unsuccessful. These data suggest the possibility of an association between congenital infections with the CVB and severe CNS defects.

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 × 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 −1 −2 + (cytolytic/suppressor) cell populations, and 2 × 10 6 of the enriched helper and cytolytic cells were adoptively transfused into infected T-cell-deficient recipients. Animals receiving the immune Lyt 2 + 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 injury in CVB3-induced myocarditis is the product of cellular immune mechanisms.

Meningo-encephalitis caused by Coxsackievirus group B type 2. Diagnosis confirmed by measuring intrathecal antibody

We describe a case of meningo-encephalitis caused by Coxsackievirus group B type 2. Diagnosis was suggested by isolation of virus from faeces and high titres of serum antibody in which Coxsackie virus B2 specific IgM antibody was detected. Evidence of intrathecal production of antibody confirmed the diagnosis.