Richard L. Crowell

Induction of neutralizing antibodies by the coxsackievirus B3 virion polypeptide, VP2

Abstract An immunogenic analysis of isolated substructures of coxsackievirus B3 virions revealed that the virion polypeptide, VP2, was the immunogen which induced neutralizing antibodies. The other substructure examined, VP1,3, elicited high antibody titers detected by enzyme-linked immunosorbent assay (ELISA), but no detectable neutralizing antibodies. Electron micrographs of the viral substructures provided evidence for ring-like structures of VP1,3 which probably serve as the basic structural matrix of coxsackievirus B3 virions. Antiserum to VP2 was shown to be monospecific by immunoprecipitation and SDS-PAGE analysis. This antiserum also precipitated native virions, indicating that VP2 is on the surface of virions. This location of VP2 is consistent with its function as immunogen and antigen in the neutralization reaction as discussed.

Indirect Enzyme-linked Immunosorbent Assay (ELISA) for the Detection of Coxsackievirus Group B Antibodies

An indirect, solid phase, microplate enzyme-linked immunosorbent assay (ELISA) was found to be highly sensitive and reliable for detecting antibodies to the group B Coxsackieviruses and other picornaviruses. Conditions for obtaining maximum sensitivity and reproducibility of the indirect ELISA are described. Antibody titres were comparable to those obtained by the virus neutralization test and over 50 times higher than those obtained by the complement-fixation test. Purified virions used in the indirect ELISA reacted with low levels of cross-reacting heterotypic antibodies elicited by each of the six group B Coxsackieviruses, although homotypic reactions resulted in highest titres.

A structural model for picornaviruses as suggested from an analysis of urea-degraded virions and procapsids of coxsackievirus B3

Abstract Urea treatment (3 M , 15 min, 37 °C, pH 9) of coxsackievirus B3 inactivated virus infectivity and degraded the virus capsid into substructures recoverable on sucrose gradients. One substructure which sedimented around 20 S contained VP1 and VP3, the other substructures which sedimented at 5 S contained VP2 and VP4, respectively, as analyzed by SDS polyacrylamide electrophoresis. The VP2 and VP4 polypeptides in the 5 S peak were probably separate since their molar ratios differed over the peak, and VP4 could be removed by dialysis. Treatment of the virions with only 1 M urea for 5 min yielded four peaks of radioactivity on sucrose gradients which sedimented at about 150 S (undegraded virions), 75–80 S (capsids minus VP4), and the 20 S and 5 S structures referred to above, suggesting a stepwise degradation of B3 virions by urea. The procapsids also were degraded into substructures which were separated on sucrose gradients; one sedimenting at around 40 S containing mostly VPO, and the other sedimenting around 20 S containing only VP1 and VP3. When coxsackievirus B3- 35 S cysteine-labeled virions were disrupted and analyzed on SDS gels, all polypeptides except VP4 were labeled, suggesting that VP2 and VP4 are distinct polypeptides. Analysis of urea-disrupted coxsackievirus B3 substructures provides the basis for a T = 3 structural model of the picornaviruses, with 12 pentamers (VP2 and VP4) and 20 hexamers (VP1 and VP3) per virion.

Immunological studies of the group B coxsackieviruses by the sandwich enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation.

A microplate double antibody sandwich ELISA was employed in an immunological study of the group B Coxsackieviruses. The assay, described in detail, detected high dilutions of virion antigen (less than 10 ng) in purified preparations and in crude infected cell extracts. Furthermore, by using a constant amount of antigen, group B virus antibodies in hamster antisera could be quantified with a sensitivity equivalent to the virus neutralization test. Titrations of virus antigens and antibodies were found to be type-specific when purified virions were employed in the assay. Urea disruption of virions exposed antigens common to all six group B viruses. The heterotypic reactivity of disrupted group B virions did not extend to the other viruses tested. Immunoprecipitation and SDS-PAGE analysis revealed that, of the four virion structural polypeptides (VPI to 4), VPI contained the major common antigenic determinants shared by members of the group B Coxsackieviruses.

A solid-phase assay of solubilized HeLa cell membrane receptors for binding group B coxsackieviruses and polioviruses

Abstract HeLa cell membranes were solubilized in sodium deoxycholate and adsorbed onto polystyrene microtiter plates to permit the measurement of receptors for binding group B Coxsackieviruses and polioviruses. Receptor activity was determined by measuring the amount of infectious virus attached to coated wells at 6°. Conditions for optimal binding of virus were determined. Receptor binding activity for group B Coxsackieviruses was detected from as few as 2.5 × 10 4 cell equivalents/ml to provide a method which was over 400 times more sensitive than the virus eclipse assay used previously. The assay revealed a recovery of up to 7% of the receptor activity of whole cells based on virus saturation of receptors. The specificity of the binding activity of the immobilized receptors was demonstrated by virus attachment competition, as done previously for receptors on live HeLa cells. Application of this method to measure receptors in deoxycholate-solubilized fetal mouse brain revealed a 2500-fold increase in sensitivity from that obtained in a virus-binding assay used previously.

Expression and distribution of the receptors for coxsackievirus B3 during fetal development of the Balb/c mouse and of their brain cells in culture

Abstract This study was designed mainly to determine the relationships between the expression and distribution of the cellular receptor proteins for coxsackievirus B3 (CVB3) and susceptibility of mouse brain cells during fetal development of Balb/c mice. Immunoblot analysis of fetal extracts demonstrated that the CVB3 receptor proteins were first expressed at day 14 of the fetal stage, and that maximal expression of the cellular receptor occurred at near term or newborn stage. Results also suggested that newborn mouse brain tissue expressed much larger quantities of viral receptor proteins, compared to other tissues. In vitro studies showed that both mouse neurons and astrocytes could be infected by two CVB3 strains, pantropic CVB3 Nancy strain (CVB3N) and myocardiotropic CVB3 Woodruff strain (CVB3W). CVB3N, however, replicated and grew to high titer in primary astrocyte cultures and in primary neuron cultures, whereas, primary astrocyte cultures were relatively resistant to CVB3W. Virus binding assays revealed that CVB3N bound faster and in greater amounts to mouse brain cells than CVBW. These two virus strains, however, were found to share the same receptor specificity by virus competition assays. The number of virus binding sites for CVB3 on newborn mouse brain cells was approximately 1.8 × 104 per cell. The data suggested that preferential expression of the cellular receptors on newborn mouse brain cells may be related to their high susceptibilities to CVB3 infection.

Receptor proteins on newborn Balb/c mouse brain cells for coxsackievirus B3 are immunologically distinct from those on HeLa cells.

Newborn Balb/c mice are highly susceptible to infection by the six coxsackievirus serotypes of group B (CVB) and it is known that receptor for these viruses are in highest concentration in the brain as compared to other tissues. Therefore, proteins from the brain tissues of these animals were solubilized (Brain-Ext) and characterized for the identification of mouse brain receptor (MBR) proteins. Virus-blot analyses of Brain-Ext suggested that each of three virus variants of CVB3-(N, W and RD) recognized four receptor proteins designated p46, p44, p36 and p33 according to their molecular size. Similar analyses of cultured neurons from newborn Balb/c mice revealed the presence of the same four receptor proteins, while astrocytes appeared to possess only p46 and/or p44. Isoelectric focusing of Brain-Ext, focused MBR proteins in the pH range 4.0-8.5, with a peak around pH 5.7. P46 was found to be neuraminidase sensitive. A polyclonal rat antiserum (anti-MBR) protected cultured neurons and astrocytes against infection by CVB3, inhibited virus binding to these cells and recognized the same four receptor proteins on western-blots as detected on virus-blots by CVB3. However, a rabbit polyclonal anti-HeLa cell antiserum, which strongly binds to HeLa cells and protects them from CVB3 infection, neither recognized any of the receptor proteins in western-blot analyses of Brain-Ext nor inhibited CVB3 infection on cultured neurons and astrocytes. Conversely, anti-MBR did not recognize any of the receptor proteins by western-blot analysis of HeLa cell extracts nor did it inhibit CVB3 infection of HeLa cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The Early Interaction of Coxsackievirus B3 with HeLa Cells

Summary Purified coxsackievirus B3 labeled with 32P- or 14C-valine interacted with HeLa cells in a manner resembling that found by others for poliovirus. Radioactivity eluted from cells at 37°, pH 7, as noninfectious virus, but contained infectious RNA. Both elution and “eclipse” of virus were inhibited irreversibly at low pH to provide further evidence that these processes may be related properties of the cell surface.

Induction of heterotypic virus resistance in adult inbred mice immunized with a variant of Coxsackievirus B3

Infection of adult male C3H/HeJ mice with a host range variant of Coxsackievirus B3 (CB3W-RD) induced resistance in these mice to an otherwise lethal dose of Coxsackievirus B1 (CB1). The protective effect induced by CB3W-RD was detectable as early as 1 day post-vaccination and was still present 10 weeks later. While untreated mice infected with CB1 died within 5 days because of massive hepatic necrosis, the liver was spared in mice immunized with CB3W-RD and then challenged with CB1. In general, CB1 titers in heart, liver, and pancreas of CB3W-RD-vaccinated animals were lower than that found in unvaccinated animals. Virus neutralizing antibody was not a mediator of this heterotypic, virus-induced protective effect. In addition, the outcome of CB1 infection could be modified if superinfection with CB3W-RD took place within 1-4 days following CB1 infection. In this regard, maximum therapeutic efficacy was observed when CB1 infected mice were superinfected 2 days after CB1 infection. CB1-infected mice that survived as a result of treatment with CB3W-RD exhibited liver regeneration but did develop myocardial necrosis.

Pathogenesis of acute myocardial necrosis in inbred mice infected with coxsackievirus B3

The pathogenesis of myocardial necrosis due to CB3W infection was studied in BALB/c and C3H/HeJ mice. BALB/c mice infected with 5 x 10(4) pfu were found to die of massive hepatic coagulative necrosis before myocardial changes occurred. Reducing the inoculum size to 5 x 10(2) pfu resulted in sublethal hepatic involvement and multifocal myocardial coagulative necrosis by day 7 p.i. In contrast, C3H/HeJ mice survived infection and developed multifocal myocardial coagulative necrosis, but not liver disease following inoculation with as much as 5 x 10(6) pfu of CB3W. As with BALB/c mice infected with 5 x 10(2) pfu, myocardial lesions became apparent in C3H/HeJ mice a few days after peak cardiac virus titer was attained. Minimal inflammatory infiltrate was seen following development of cellular necrosis and was restricted to the areas of virus-induced pathologic change. However, no evidence was found for virus-specific cytotoxic T cell activity or for delayed type hypersensitivity responses. Furthermore, myocardial necrosis in CB3W-infected, T cell-depleted C3H/HeJ mice was as severe as in CB3W-infected, immunocompetent mice. These data have led us to conclude that cardiac lesions were due to virus-induced cytopathology rather than immunopathogenic mechanisms.