Kisoon Kim

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.

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.

Enteroviruses, type 1 diabetes and hygiene: a complex relationship

Type 1 diabetes (T1D) is an autoimmune disease in which the immune system mounts an attack on the hosts insulin‐producing β cells. Because most cases of T1D cannot be attributed only to individual genetics, it is strongly inferred that there is a significant environmental contribution, such as infection, impacting disease development. The human enteroviruses (HEV) are common picornaviruses often implicated as triggers of human T1D, although precisely which of the numerous HEV may be involved in human T1D development is unknown. Experiments using non‐obese diabetic (NOD) mice, commonly used to model T1D, show that induction of T1D by HEV infection in NOD mice is a multifactorial process involving both the virus and the host. Interestingly, results demonstrate that HEV infection of NOD mice can also induce long‐term protection from T1D under certain conditions, suggesting that a similar mechanism may occur in humans. Based upon both experimental animal and observational human studies, we postulate that HEV have a dual role in T1D development and can either cause or prevent autoimmune disease. Whichever outcome occurs depends upon multiple variables in the host‐virus equation, many of which can be deduced from results obtained from NOD mouse studies. We propose that the background to the sharply rising T1D incidences observed in the 20th century correlates with increased levels of hygiene in human societies. Viewing T1D in this perspective suggests that potential preventative options could be developed. Copyright

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.

Complete Genome Sequence of Human Respiratory Syncytial Virus Genotype A with a 72-Nucleotide Duplication in the Attachment Protein G Gene

ABSTRACT The complete genome sequence of human respiratory syncytial virus genotype A (HRSV-A) with a 72-nucleotide duplication in the C-terminal part of the attachment protein G gene was determined and analyzed. The genome was 15,277 bp in length, and 0.46 to 6.03% variations were identified at the nucleotide level compared with the previously reported complete genome of HRSV-A. Characterization of the genome will improve understanding of the diversity of the HRSV-A major antigens and enable an in-depth analysis of its genetics.

Rapid replacement of human respiratory syncytial virus A with the ON1 genotype having 72 nucleotide duplication in G gene

Abstract Human respiratory syncytial virus (HRSV) is the main cause of severe respiratory illness in young children and elderly people. We investigated the genetic characteristics of the circulating HRSV subgroup A (HRSV-A) to determine the distribution of genotype ON1, which has a 72-nucleotide duplication in attachment G gene. We obtained 456 HRSV-A positive samples between October 2008 and February 2013, which were subjected to sequence analysis. The first ON1 genotype was discovered in August 2011 and 273 samples were identified as ON1 up to February 2013. The prevalence of the ON1 genotype increased rapidly from 17.4% in 2011–2012 to 94.6% in 2012–2013. The mean evolutionary rate of G protein was calculated as 3.275×10−3 nucleotide substitution/site/year and several positively selected sites for amino acid substitutions were located in the predicted epitope region. This basic and important information may facilitate a better understanding of HRSV epidemiology and evolution.

Replication of Coxsackievirus B3 in Primary Cell Cultures Generates Novel Viral Genome Deletions

ABSTRACT Coxsackievirus B3 (CVB3) generates 5′-terminally deleted genomes (TDs) during replication in murine hearts. We show here that CVB3 populations with TDs can also be generated within two to three passages of CVB3 in primary, but not immortalized, cell cultures. Deletions of less than 49 nucleotides increase in size during passage, while 5′ TDs of 49 nucleotides appear to be the maximum deletion size. The cellular environment of contact-inhibited primary cell cultures or the myocardium in vivo is sufficient for the selection of 5′ TDs over undeleted genomes.

Coxsackievirus Expression of the Murine Secretory Protein Interleukin-4 Induces Increased Synthesis of Immunoglobulin G1 in Mice

ABSTRACT We cloned the sequence encoding murine interleukin-4 (mIL-4), including the secretory signal, into the genome of CVB3/0, an artificially attenuated strain of coxsackievirus B3, at the junction of the capsid protein 1D and the viral protease 2Apro. Two strains of chimeric CVB3 were constructed using, in one case, identical sequences to encode 2Apro cleavage sites (CVB3/0-mIL4/47) on either side of the inserted coding sequence and, in the other case, nonidentical sequences that varied at the nucleotide level without changing the amino acid sequences (CVB3-PL2-mIL4/46). Transfection of HeLa cells yielded progeny viruses that replicated with rates similar to that of the parental CVB3/0 strain, although yields of mIL-4-expressing strains were approximately 10-fold lower than those of the parental virus. Western blot analysis of viral proteins isolated from HeLa cells inoculated with either strain of chimeric virus demonstrated that the chimeric viruses synthesized capsid protein 1D at approximately twofold-higher levels than the parental virus. mIL-4 protein was detected by enzyme-linked immunosorbent assay (ELISA) in HeLa cells inoculated with either strain of chimeric virus. Lysates of HeLa cells inoculated with either chimeric virus induced the proliferation of the mIL-4-requiring murine MC-9 cell line, demonstrating biological activity of the CVB3-expressed mIL-4. Reverse transcription (RT)-PCR analysis of viral RNA derived from sequential passaging of CVB3/0-mIL4/47 in HeLa cells demonstrated deletion of the mIL-4 coding sequence occurring by the fourth passage, while similar analysis of CVB3-PL2-mIL4/46 RNA demonstrated detection of the mIL-4 coding sequence in the virus population through 10 generations in HeLa cells. mIL-4 protein levels determined by ELISA were consistent with the stability and loss data determined by RT-PCR analysis of the passaged viral genomes. Studies of insert stability of CVB3-PL2-mIL4/46 during replication in mice showed the presence of the viral mIL-4 insert in pancreas, heart, and liver at 14 days postinfection. Comparison of the murine antibody responses to CVB3-PL2-mIL4/46 and the parental CVB3/0 strain demonstrated an increased level of CVB3-binding serum immunoglobulin G1 in mice inoculated with CVB3-PL2-mIL4/46.

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.

Genetic determinants of virulence in the group B coxsackieviruses

The group B coxsackieviruses (CVB) are well-studied human enteroviruses that are established causes of numerous serious human diseases. Characterized differences in CVB genomes of different strains affect the ability with which specific strains induce disease in the mouse host and, by inference, in humans as well. The first hurdle is to define specific examples of CVB genetic changes that are associated with pathogenic phenotypes. Such differences have been mapped both to coding and noncoding genomic regions. Many studies have used laboratory-derived strains to identify genetic differences that are essential to phenotype expression, work that is valuable but requires confirmation from studies of wild-type isolates. Rapid viral replication is closely associated with acute disease, indicating a key role for viral damage to the host, while host-mediated responses to the viral infection and viral persistence over a longer period of time indicate other roles for the virus in pathogenesis.