Nora M. Chapman

Toward Testing the Hypothesis that Group B Coxsackieviruses (CVB) Trigger Insulin-Dependent Diabetes: Inoculating Nonobese Diabetic Mice with CVB Markedly Lowers Diabetes Incidence

ABSTRACT Insulin-dependent (type 1) diabetes mellitus (T1D) onset is mediated by individual human genetics as well as undefined environmental influences such as viral infections. The group B coxsackieviruses (CVB) are commonly named as putative T1D-inducing agents. We studied CVB replication in nonobese diabetic (NOD) mice to assess how infection by diverse CVB strains affected T1D incidence in a model of human T1D. Inoculation of 4- or 8-week-old NOD mice with any of nine different CVB strains significantly reduced the incidence of T1D by 2- to 10-fold over a 10-month period relative to T1D incidences in mock-infected control mice. Greater protection was conferred by more-pathogenic CVB strains relative to less-virulent or avirulent strains. Two CVB3 strains were employed to further explore the relationship of CVB virulence phenotypes to T1D onset and incidence: a pathogenic strain (CVB3/M) and a nonvirulent strain (CVB3/GA). CVB3/M replicated to four- to fivefold-higher titers than CVB3/GA in the pancreas and induced widespread pancreatitis, whereas CVB3/GA induced no pancreatitis. Apoptotic nuclei were detected by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay in CVB3/M-infected pancreata but not in CVB3/GA-infected pancreata. In situ hybridization detected CVB3 RNA in acinar tissue but not in pancreatic islets. Although islets demonstrated inflammatory infiltrates in CVB3-protected mice, insulin remained detectable by immunohistochemistry in these islets but not in those from diabetic mice. Enzyme-linked immunosorbent assay-based examination of murine sera for immunoglobulin G1 (IgG1) and IgG2a immunoreactivity against diabetic autoantigens insulin and HSP60 revealed no statistically significant relationship between CVB3-protected mice or diabetic mice and specific autoimmunity. However, when pooled sera from CVB3/M-protected mice were used to probe a Western blot of pancreatic proteins, numerous proteins were detected, whereas only one band was detected by sera from CVB3/GA-protected mice. No proteins were detected by sera from diabetic or normal mice. Cumulatively, these data do not support the hypothesis that CVB are causative agents of T1D. To the contrary, CVB infections provide significant protection from T1D onset in NOD mice. Possible mechanisms by which this virus-induced protection may occur are discussed.

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.

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

ABSTRACT 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.

The diagnosis of insulitis in human type 1 diabetes

To the Editor: During a workshop concerning the histopathological characteristics of insulitis in human type 1 diabetes (fifth annual meeting of the JDRF Network for Pancreatic Organ Donors with Diabetes [nPOD], 10 February 2013, Jacksonville, FL, USA), a consensus opinion was reached on the criteria necessary for the diagnosis of insulitis, and a definition of insulitis was agreed, as detailed in the text box. Workshop attendees included many leading researchers in the

Persistent coxsackievirus infection: enterovirus persistence in chronic myocarditis and dilated cardiomyopathy.

Enteroviral infection of the heart has been noted in a significant proportion of cases of myocarditis and dilated cardiomyopathy. The presence of enterovirus RNA at stages of disease after acute infection and correlation of enterovirus replication with worse clinical outcome suggests continued replication of the virus is involved in the progression of the disease. This finding is mirrored by the murine model of coxsackievirus B3 myocarditis, in which virus persists through the evolution of the virus to a terminally deleted defective form which persists in the myocardium. Studies of the mechanism of induction of myocarditis by coxsackievirus B3 require assessment of the effects of alterations of the immune response upon virus persistence in this form. As expression of viral proteins in the heart have been shown to generate significant impairment of cardiomyocyte function and promote generation of dilated cardiomyopathy, the role of virus persistence is likely to include direct effects of viral replication as well as induction of inflammation in the heart. Factors that control the extent of cardiac infection with terminally deleted enteroviruses and the relative roles of continued immune response of the virus vs viral modification of cardiac function need to be measured to find effective therapies for the human disease.

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.

Coxsackievirus B3 from an infectious cDNA copy of the genome is cardiovirulent in mice.

SummaryA coxsackievirus B3 (CVB3) cDNA clone, upon transfection of HeLa cells, produces CVB3 capable of induction of cardiac inflammation in C3H/He mice by day 8 post inoculation (p.i.). Liver and serum are cleared of CVB3 by day 8 p.i., but CVB3 persists in the heart through day 14. The nucleotide sequence and the predicted amino acid sequence of this clone have sites of divergence from 2 other completely sequenced CVB3 genomes although overall identity of the three CVB3 genomes is 99%.

Group B coxsackievirus diabetogenic phenotype correlates with replication efficiency

ABSTRACT Group B coxsackieviruses can initiate rapid onset type 1 diabetes (T1D) in old nonobese diabetic (NOD) mice. Inoculating high doses of poorly pathogenic CVB3/GA per mouse initiated rapid onset T1D. Viral protein was detectable in islets shortly after inoculation in association with beta cells as well as other primary islet cell types. The virulent strain CVB3/28 replicated to higher titers more rapidly than CVB3/GA in the pancreas and in established beta cell cultures. Exchange of 5′-nontranslated regions between the two CVB3 strains demonstrated a variable impact on replication in beta cell cultures and suppression of in vivo replication for both strains. While any CVB strain may be able to induce T1D in prediabetic NOD mice, T1D onset is linked both to the viral replication rate and infectious dose.

A Group B Coxsackievirus/Poliovirus 5′ Nontranslated Region Chimera Can Act as an Attenuated Vaccine Strain in Mice

ABSTRACT The linear, single-stranded enterovirus RNA genome is flanked at either end with a nontranslated region (NTR). By replacing the entire 5′ NTR of coxsackievirus B3 (CVB3) with that from type 1 poliovirus, a progeny virus was obtained following transfection of HeLa cells. The chimeric virus, CPV/49, replicates like the parental CVB3 strain in HeLa cells but is attenuated for replication and yield in primary human coronary artery endothelial cell cultures, in a human pancreas tumor cell line, and in primary murine heart fibroblast cultures. Western blotting analyses of CPV/49 replication in murine heart fibroblast cultures demonstrate that synthesis of CPV/49 proteins is significantly slower than that of the parental CVB3 strain. CPV/49 replicates in murine hearts and pancreata, causing no disease in hearts and a minor pancreatic inflammation in some mice that resolves by 28 days postinoculation. A single inoculation with CPV/49 induces protective anti-CVB3 neutralizing antibody titers that completely protect mice from both heart and pancreatic disease when mice are challenged 28 days p.i. with genetically diverse virulent strains of CVB3. That a chimeric CVB3 strain, created from sequences of two virulent viruses, is sufficiently attenuated to act as an avirulent, protective vaccine strain in mice suggests that chimeric genome technology merits further evaluation for the development of new nonpoliovirus enteroviral vectors.