Charles J. Gauntt

An infectious cDNA copy of the genome of a non-cardiovirulent coxsackievirus B3 strain : its complete sequence analysis and comparison to the genomes of cardiovirulent coxsackieviruses

SummaryThe genome of the non-cardiovirulent coxsackievirus B3 (CVB3) strain CVB3/0 was cloned and sequenced to aid in the elucidation of the viral genetic basis for the CVB3 cardiovirulent phenotype. Reverse-transcribed sub-genomic complementary DNA (cDNA) fragments were enzymatically amplified using generic oligonucleotide primers and were assembled as a complete infectious genomic copy (pCVB3-0) downstream of the T7 RNA polymerase promoter. Positive-strand viral RNA transcribed from pCVB3-0 using T7 RNA polymerase and transfected into HeLa cells produced infectious virus (CVB3/0c). No differences in phenotype were observed comparing growth of CVB3/0c to the parental CVB3/0 in HeLa single-step growth curves, virus yields, or plaque size. When inoculated into C3H/HeJ mice. CVB3/0c achieved cardiac titers equivalent to the parental CVB3/0 and like the parental virus, demonstrated a non-cardiovirulent phenotype. The nucleotide sequence of the cloned CVB3/0 genome was determined and compared to the genomes of infectious cDNA clones of cardiovirulent CVB3 strains. Two consistent differences among nucleotides in non-translated regions and 8 amino acid differences relative to two well-characterized infectious cDNA copies of genomes from cardiovirulent CVB3 strains were identified.

Molecular approaches to enteroviral diagnosis in idiopathic cardiomyopathy and myocarditis

Enteroviruses are thought to be etiologic agents in some cases of human myocarditis and dilated cardiomyopathy. Murine models of acute coxsackievirus B3 myocarditis implicate coxsackie B viruses as possible causes of human myocarditis. Indirect evidence implicating enteroviruses as causative agents in human heart disease derives from serologic studies. More recently, direct evidence for enteroviral presence in diseased human heart tissues has been obtained by nucleic acid hybridization analyses. Although the data suggest that enteroviral infections may be associated with 18% to 50% of cases of myocarditis or dilated cardiomyopathy, or both, causality has not been established. Unanswered questions remain regarding the specific identity of the enteroviral genomes detected in the human heart and the potential for enteroviruses to persist in the heart.

Group B coxsackievirus myocarditis and pancreatitis: Connection between viral virulence phenotypes in mice

The group B coxsackieviruses (CVB) induce experimental pancreatitis and myocarditis in mice and are established agents of human myocarditis, especially in children. We tested the hypothesis that the development of CVB‐induced myocarditis is linked to CVB‐induced pancreatitis by studying the replication of different CVB strains in mice. Eight of nine genotypically different type 3 CVB (CVB3) strains induced acute pancreatitis in mice; of these, three viruses also induced acute myocarditis. One CVB3 strain was avirulent for both organs. Myocarditis was not observed in the absence of pancreatitis. The results obtained by inoculation of mice with strains of other CVB serotypes were consistent with these data. Infectious virus titers were measured in serum, pancreas, and heart as a function of time after inoculation of mice with three CVB3 strains. Each strain was representative of one of the three viral virulence phenotypes: avirulent, pancreovirulent only, and cardiovirulent. All strains replicated well and persisted in the pancreas through 8 days post‐inoculation, but the cardiovirulent CVB3 strain tended to replicate to higher titer earlier and persist longer in sera, pancreatic, and cardiac tissues than the noncardiovirulent strains. Replication of the CVB3 strains were studied in two human pancreatic tumor lines and in primary human endothelial cell cultures derived from cardiac artery. Cardiovirulent strains, both individually and as a group, tended to replicate to titers as high as, or higher than, noncardiovirulent strains did in cell culture. The data are consistent with the possibility of an etiologic link between CVB‐induced pancreatic and heart disease. J. Med. Virol. 62:70–81, 2000.

Role of natural killer cells in experimental murine myocarditis

ConclusionsInfection of adolescent CD-1 mice with CVB3 activates NK cells. These activated NK cells can lyse infected fibroblasts in vitro and their transfer into mice prior to inoculation with CVB3 can reduce virus titers in heart tissues. If mice are depleted of NK cells prior to and throughout a CVB3 infection, myocarditic lesions show a comparatively greater pathology in the form of severe dystrophic calcification than in infected mice with an intact NK cell system. Also NK cell-depleted mice have significantly higher virus titers in heart tissues. These data suggest a role for NK cells in the infected mouse, i. e., that they reduce virus titers through lysis of infected cells and thereby reduce the severity of myocarditis. In keeping with such a defensive role is the finding of NK cells among the first inflammatory cells forming the nascent CVB3-induced focal myocarditic lesion. Paradoxically, NK cells remain in the mature lesion up to 10 days p.i. and may thus contribute to pathology. Further studies on whether these long-term residents release cytotoxic factors which continue to damage myocytes in absence of significant virus titers in heart tissues must be performed. These studies are now feasible because of the recent development of monoclonal antibodies against NK cytotoxic factor [30]. Finally, diet can adversely effect generation of activated NK cells in CVB3-inoculated mice and this finding may have significance for health-conscious human beings.

The Possible Role of Viral Variants in Pathogenesis

Many different kinds of coxsackievirus (CV) variants have been described and studied with the goal of trying to relate a set of characteristics of a given variant to its pathogenicity in cell cultures or in animal models. In reading about studies about a particular variant, one must remember that any virus stock represents a genotypically heterogeneous population from which phenotypic expression of a dominant variant(s) is observed and measured. Murine models of CV diseases offer exciting possibilities for determining the molecular basis of virulence in these viruses.

High-efficiency polyethylene glycol-mediated transformation of mammalian cells.

A new, high-efficiency method for transformation of mammalian cells with nucleic acids is described which yields 105−106 plaques/μg poliovirus infectious RNA (iRNA). The optimized procedure consists of two steps: (1) exposure of cells to iRNA in a high ionic-strength buffer followed by (2) a brief exposure to a 35% polyethylene glycol (PEG) solution. Optimized conditions for each variable in the procedure are described. Under optimized conditions for PEG-mediated transformation with RNA, large numbers of transformants are recovered with plasmid DNA as well. The procedure presented is similar to other high-efficiency PEG-mediated methods previously described for the genetic transformation of both nonprotoplasted Escherichia coliand yeast.

Progress toward vaccines against viruses that cause heart disease.

Of the numerous viruses that have been implicated as causes of viral inflammatory cardiomyopathy, only the 6 serotypes of the group B coxsackieviruses (CVB 1–,6) and adenovirus type 2 (Ad2) have been regularly linked to heart disease on the basis of both clinical investigations as well as animal models (in the case of the coxsackieviruses). Of these, only the coxsackieviruses offer a truly well-characterized system for not only investigations using a small animal disease model (myocarditis in mice) but for studies of the virus at the molecular level and in cell culture systems. The pending worldwide eradication of the related enteroviruses, the polioviruses, will further emphasize the importance of the coxsackieviruses in years to come. Studies using poliovirus have shown that enteroviruses can be attenuated for disease to create highly successful and safe human vaccines. Furthermore, using recombinant DNA approaches, strains of polioviruses have been created that demonstrate a human enterovirus can express small proteins as well as foreign antigenic epitopes, thus creating multivalent chimeric vaccine strains of virus, Our laboratory has been exploring coxsackievirus 3-based vectors as models for both multivalent chimeric vaccines as well as expression vectors. The coxsackievirus can also express intact small proteins in biologically active form as well as antigenic epitopes. Although it is doubtful that the marketplace will support the development of antiviral vaccines to combat human heart disease at present, the technology exists to make such vaccines a reality.ZusammenfassungVon den zahlreichen Viren, die als mögliche Ursache viraler entzündlicher Kardiomyopathien diskutiert wurden, konnte bisher nur für die sechs Serotypen der Gruppe-B-Coxsackie-Viren (CVB 1–6) und für das Adenovirus Typ 2 (Ad 2), basierend auf klinischen Studien und im Fall der Coxsackie-Viren in Tiermodellen, deren regelmäßige Verbindung zu Herzerkrankungen gezeigt werden. Insbesondere Coxsackie-Viren stellen ein gut charakterisiertes System dar, sowohl für Studien im Kleintiermodell (Myokarditis in Mäusen) als auch für Untersuchungen auf molekularer Ebene und in Zellkultur. Die anstehende weltweite Eradikation der verwandten Enteroviren, der Polioviren, betont die weitere zukünftige Bedeutung der Coxsackie-Viren. Untersuchungen mit Polioviren konnten zeigen, daß Enteroviren in einer abgeschwächten Form eine äußerst wirkungsvollen und sicheren Impfstoff darstellen können. Des weiteren sind unter Verwendung rekombinanter DNA-Techniken Poliovirusstämme entwickelt worden, mit denen gezeigt werden konnte, daß ein humanes Enterovirus sowohl kleinere Proteine als auch antigene Fremdepitope exprimieren und somit einen multivalenten rekombinanten Virusstamm darstellen kann.Unsere Arbeitsgruppe hat auf Coxsackie-Virus 3 basierende Vektoren als Modell für multivalente chimere Impfstoffe als auch als Expressionsvektoren untersucht. Coxsackie-Viren können durch Punktmutationen und mit Hilfe von rekombinanter Gentechnologie erfolgreich attenuiert werden. Coxsackie-Viren können darüber hinaus biologisch aktive, kleine Proteine sowie antigene Epitope exprimieren. Obwohl es zweifelhaft ist, daß die Wirtschaft die Entwicklung von antiviralen Impfstoffen zur Bekämpfung von Herzerkrankungen zum jetzigen Zeitpunkt unterstützen wird, ist die Technologie, solche Impfstoffe Realität werden zu lassen, bereits verfügbar.

Early Cellular Infiltrates in Coxsackievirus B3 Murine Myocarditis

The pathologic basis of murine myocarditis induced by coxsackievirus type B3 (CVB3) places necessary emphasis on the inflammatory infiltrate [11, 21]. In efforts to establish the phenotypic identity of infiltrating cells, investigators have used a variety of methods, including selective depletion or enrichment of defined cellular subsets [5, 10, 16], semiquantitative in situ immune autoradiography [8], immuno-electron microscopy [3], and immunohistochemistry [4, 19]. Such studies, however, have yet to provide serially comprehensive and quantitative data pertinent to the evolution of the inflammatory response in the earliest phase of the disease. Better understanding of the way in which phenotypically different subpopulations of immune cells invade the myocardium may help to explain the potential genesis of autoimmunity inducible in various strains of mice infected with CVB3 [1, 13]. The ever-expanding complement of monoclonal antibodies directed against cell surface antigens, with greater and greater specificity for cell type and state of activation, permits progressively more distinctive characterization.

Fragility of the Rhinovirus Type 14 Genome to Incubation at 60

Rhinovirus type 14 (RV14) genomic RNA, but not mengovirus or coxsackievirus B3 genomic RNA, fragmented to acid-insoluble RNA species during incubation in aqueous buffers at 60 degrees. Incubation of RV14 genomic RNA in high concentrations of dimethyl sulfoxide at 60 degrees did not result in fragmentation, suggesting that fragmentation was not a result of pre-existing nicks. RV14RNA that was treated with proteinase K prior to incubation at 60 degrees did not undergo fragmentation. The association of protein(s) with RV14 virion RNA was demonstrated by radiolabeling with amino acids. These results showed that protein(s) associated with RV14 genomic RNA was in some manner responsible for fragmentation of the RNA during incubation at 60 degrees.

Actinomycin D acts on an intracellular process to inhibit replication of rhinovirus type 14.

Summary Act D inhibited replication of RV14 but not mengovirus in HeLa cells. Early processes such as attachment, penetration, and uncoating of RV14 were not affected by Act D. Pretreatment of cells with Act D and removal of the antibiotic prior to infection increased the antiviral effect of Act D on replication of RV14. Yields of RV14 per cell were reduced in Act D-pretreated cell cultures but the number of virus-producing cells was not reduced. Cells treated with Act D for 6 hr prior to inoculation of RV14 synthesized only 28% of the ribonuclease-sensitive and ribonuclease-re-sistant RV14 RNA which was synthesized by cells treated with Act D at the time of virus inoculation. As most ribonuclease-re-sistant RNA is associated with viral replicative-intermediate RNA structures in these cells, the results are compatible with the hypothesis that a cell process is required for synthesis of viral replicative-intermediate RNA and it is the cell process with which Act D interferes and contributes to the reduction in RV14 yields. I thank David R. L. LaBadie for conscientious and excellent technical assistance. This study was funded in part by an American Heart Association, Texas Affiliate, Inc. grant and by an Institutional Research grant from the University of Texas Health Science Center at San Antonio. I thank Kendall O. Smith and Stephen J. Mattingly for constructive criticisms of the manuscript.