The extrachromosomal elements of the Naegleria genus: How little we know.

There are currently 47 characterized species in the Naegleria genus of free-living amoebae. Each amoeba has thousands of extrachromosomal elements that are closed circular structures comprised of a single ribosomal DNA (rDNA) copy and a large non-rDNA sequence. Despite the presence of putative open reading frames and introns, ribosomal RNA is the only established transcript. A single origin of DNA replication (ori) has been mapped within the non-rDNA sequence for one species (N. gruberi), a finding that strongly indicates that these episomes replicate independently of the cell's chromosomal DNA component. This article reviews that which has been published about these interesting DNA elements and by analyzing available sequence data, discusses the possibility that different phylogenetically related clusters of Naegleria species individually conserve ori structures and suggests where the rRNA promoter and termination sites may be located.

Domain I of the 5' non-translated genomic region in coxsackievirus B3 RNA is not required for productive replication.

Domain I is a cloverleaf-like secondary structure at the 5' termini of all enterovirus genomes, comprising part of a cis-acting replication element essential for efficient enteroviral replication. 5' genomic terminal deletions up to as much as 55% of domain I can occur without lethality following coxsackie B virus infections. We report here that the entire CVB structural domain I can be deleted without lethality.

Mutational Disruption of cis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5'-Terminal Genomic Deletions.

UNLABELLED: Following natural human or experimental murine infections and in cell culture, coxsackievirus B (CVB) RNA can persist for weeks in the absence of a cytopathic effect, yet viral RNA remains detectable. Our earlier studies demonstrated that this persistence produced viral RNA with up to 49 nucleotide deletions at the genomic 5' terminus which partially degraded the cloverleaf (or domain I), an RNA structure required for efficient viral replication. A cis-acting replication element (CRE) in the 2C protein-coding region [CRE(2C)] templates the addition of two uridine residues to the virus genome-encoded RNA replication primer VPg prior to positive-strand synthesis. Because our previous work also demonstrated that the genomes of CVB with a 5'-terminal deletion (CVB-TD) have VPg covalently linked, even though they rarely terminate in the canonical UU donated by CRE(2C)-mediated uridylylation of VPg, we hypothesized that a functional (uridylylating) CRE(2C) would be unnecessary for CVB-TD replication. Using the same 16 mutations in the CVB3 CRE(2C) structure that were considered lethal for this virus by others, we demonstrate here both in infected cell cultures and in mice that wild-type (wt) and CVB3-TD strains carrying these mutations with a nonuridylylating CRE(2C) are viable. While the wt genome with the mutated CRE(2C) displays suppressed replication levels similar to those observed in a CVB3-TD strain, mutation of the CRE(2C) function in a CVB3-TD strain does not further decrease replication. Finally, we show that replication of the parental CVB3 strain containing the mutated CRE(2C) drives the de novo generation of genomic deletions at the 5' terminus. IMPORTANCE: In this report, we demonstrate that while CVB can replicate without a uridylylating CRE(2C), the replication rate suffers significantly. Further, deletions at the 5' terminus of the genome are generated in this virus population, with this virus population supplanting the wild-type population. This demonstrates that VPg can prime without being specifically uridylylated and that this priming is error prone, resulting in the loss of sequence information from the 5' terminus. These findings have significance when considering the replication of human enteroviruses, and we believe that these data are unattainable in a cell-free system due to the poor replication of these CRE-deficient viruses.

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.

Replication of coxsackievirus B3 in primary cell cultures generates novel viral genome deletions.

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.

Evolution of virulence in picornaviruses.

The Picornaviridae encompass many positive-strand RNA viruses, all of which share a generally similar genome design and capsid structure, but which induce quite diverse diseases in humans and other animals. Picornavirus strains of the same serotype have been shown to express different virulence (or pathogenic) phenotypes when studied in animal models, demonstrating that key elements of pathogenesis reside in the viral genome. However, the genetics that determine the virulence phenotype of any picornavirus are poorly understood. Picornaviruses do not have virulence genes per se, but the design ofthe capsid andhow it interacts with the virus receptor expressed on the host cell surface, specific sequences within the nontranslated regions of the viral genome, as well as coding sequences that result in different protein sequences may all have a part in determining the virulence phenotype. Virulence may be better understood as a continuum from an apparent inability to induce disease to the ability to cause severe pathogenic changes. Ultimately, the ability of a picornavirus to induce disease depends upon viral genetics and how they are modulated by the host environment.

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.

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.

Characterization of an infectious cDNA copy of the genome of a naturally occurring, avirulent coxsackievirus B3 clinical isolate.

Group B coxsackieviruses (CVB) cause numerous diseases, including myocarditis, pancreatitis, aseptic meningitis and possibly type 1 diabetes. To date, infectious cDNA copies of CVB type 3 (CVB3) genomes have all been derived from pathogenic virus strains. An infectious cDNA copy of the well-characterized, non-pathogenic CVB3 strain GA genome was cloned in order to facilitate mapping of the CVB genes that influence expression of a virulence phenotype. Comparison of the sequence of the parental CVB3/GA population, derived by direct RT-PCR-mediated sequence analysis, to that of the infectious CVB3/GA progeny genome demonstrated that an authentic copy was cloned; numerous differences were observed in coding and non-coding sequences relative to other CVB3 strains. Progeny CVB3/GA replicated similarly to the parental strain in three different cell cultures and was avirulent when inoculated into mice, causing neither pancreatitis nor myocarditis. Inoculation of mice with CVB3/GA protected mice completely against myocarditis and pancreatitis induced by cardiovirulent CVB3 challenge. The secondary structure predicted for the CVB3/GA domain II, a region within the 5' non-translated region that is implicated as a key site affecting the expression of a cardiovirulent phenotype, differs from those predicted for cardiovirulent and pancreovirulent CVB3 strains. This is the first report characterizing a cloned CVB3 genome from an avirulent strain.

Toward testing the hypothesis that group B coxsackieviruses (CVB) trigger insulin-dependent diabetes: inoculating nonobese diabetic mice with CVB markedly lowers diabetes incidence.

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.

The group B coxsackieviruses and myocarditis.

The six serotypes of the group B coxsackieviruses (CVB) are common human enteroviruses linked etiologically to inflammatory cardiomyopathies. This has been demonstrated by molecular detection of enteroviral RNA in human heart tissue, serologic associations with disease, and virus isolation from cases of fulminant myocarditis. The murine model of CVB-associated myocarditis has demonstrated that CVB can be attenuated through mutations at different genomic sites. Human CVB3 isolates demonstrate varying degrees of cardiovirulence in the murine model; one site of virulence determination has been mapped to domain II of the 5' non-translated region. The interplay of CVB replication and the immune response to that replication in the heart is a complex interaction determining the extent to which the virus replication is limited and the degree to which a pathogenic inflammation of cardiac muscle occurs. Studies of CVB3-induced myocarditis in murine strains lacking subsets of the immune system or genes regulating the immune response have demonstrated a pivotal role of the T cell response to the generation of myocarditis. While CVB are associated with 20-25% of cases of myocarditis or cardiomyopathy, the severity of the disease and the existence of attenuated strains shown to generate protective immunity in animal models indicates that vaccination against the CVBs would be valuable.

Coxsackievirus and adenovirus receptor (CAR) binds immunoglobulins.

The coxsackievirus and adenovirus receptor protein (CAR) serves as the cell surface receptor for group B coxsackieviruses and most adenoviruses, but the physiological function and ligand for this protein remain to be described. An affinity column was constructed with the recombinant extracellular domain of the CAR (rECAR) to isolate potential ligands by affinity chromatography. Immunoglobulins G and M were consistently isolated from human sera passed through the column, suggesting that the CAR may be an immunoglobulin-binding protein. Further investigation revealed that the affinity-purified immunoglobulins bound to rECAR-coated immunoassay plates, and the peroxidase-labeled rECAR bound the immunoglobulins on ligand-overlay blots. The peroxidase-labeled rECAR was incorporated into immunoprecipitates formed between the affinity-purified immunoglobulins and rabbit antibodies against human immunoglobulins, but not into immunoprecipitates formed between mouse IgG and rabbit antibodies against mouse IgG. The CAR present in HeLa cell lysates also bound to the affinity-purified immunoglobulins on Immobilon membranes, showing that the association is not limited to the recombinant protein. These results demonstrate that the CAR binds IgG and IgM present in serum, and reveal a direct interaction between the coxsackievirus and adenovirus receptor and the immune system.

Coxsackievirus expression of the murine secretory protein interleukin-4 induces increased synthesis of immunoglobulin G1 in mice.

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 (Ad 2) 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 be successfully attenuated using both point mutations as well as chimeric genome technology. 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.

Genomic determinants of cardiovirulence in coxsackievirus B3 clinical isolates: localization to the 5' nontranslated region.

Coxsackievirus B3 (CVB3) infections can cause myocarditis in humans and are implicated in the pathogenesis of dilated cardiomyopathy. The natural genetic determinants of cardiovirulence for CVB3 have not been identified, although using strains engineered in the laboratory, cardiovirulence determinants have been identified in the CVB3 5' nontranslated region (5'NTR) and capsid. The myocarditic phenotypes of two CVB3 clinical isolates were determined using an established murine model of inflammatory heart disease. The 5'NTRs and capsid proteins of the noncardiovirulent CVB3/CO strain and cardiovirulent CVB3/AS strain were examined to determine their influence on the cardiovirulence phenotype. Six intratypic chimeric viruses were constructed in which 5'NTR and capsid sequences of the infectious cDNA copy of the cardiovirulent CVB3/20 genome were replaced by homologous sequences from CVB3/CO or CVB3/AS. Chimeric strains were tested for cardiovirulence by inoculation of C3H/HeJ mice. Sections of hearts removed at 10 days postinoculation were examined for evidence of myocarditis by light microscopy and assayed for the presence of virus. Replacement of the CVB3/20 capsid coding region by that from the homologous region of CVB3/CO resulted in no change in the cardiovirulent CVB3/20 phenotype, with virus recoverable from the heart at 10 days postinoculation. However, recombinant virus containing the CVB3/CO 5'NTR alone or the 5'NTR and capsid sequences together were not myocarditic, and infectious virus was not recovered from the myocardium. Chimeric viruses containing the CVB3/AS 5'NTR alone, capsid sequence alone, or both together preserved the myocarditic phenotype. These data support the 5'NTR as the primary site in the determination of the natural cardiovirulence phenotype of CVB3.

A group B coxsackievirus/poliovirus 5' nontranslated region chimera can act as an attenuated vaccine strain in mice.

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.

Expression of the coxsackievirus and adenovirus receptor in cultured human umbilical vein endothelial cells: regulation in response to cell density.

Primary cultures of human umbilical vein endothelial cells (HUVEC) express the human coxsackievirus and adenovirus receptor (HCAR). Whereas HCAR expression in HeLa cells was constant with respect to cell density, HCAR expression in HUVEC increased with culture confluence. HCAR expression in HUVEC was not quantitatively altered by infection with coxsackievirus B.

Sites other than nucleotide 234 determine cardiovirulence in natural isolates of coxsackievirus B3.

The genetic site(s) that naturally determine the cardiovirulence phenotype of coxsackievirus B3 (CVB3) have yet to be mapped. Using two closely related CVB3 strains that differed in terms of cardiovirulence phenotype in mice, we previously reported the difference in phenotype mapped to a single site, nucleotide 234 (nt234) in the 5' non-translated region (NTR) of the CVB3 genome. When nt234 was C, the virus was attenuated and when U, the virus was cardiovirulent. To determine whether this finding was applicable to other strains of CVB3, we examined 13 different naturally occurring CVB3 strains isolated in different years in the United States. We determined that only two isolates induced severe inflammatory heart muscle disease in C3H/HeJ male mice. Using PCR products as sequencing templates, we determined the 5' NTR sequence from each viral genome. Alignment of these sequences and other published CVB3 5' NTR sequences suggests as many as four separate lineages, with commonly used laboratory strains clustering closely in one branch. An examination of the sequences showed that regardless of cardiovirulence phenotype, nt234 was invariably uridine. Thus, the previously reported cytidine at nt234 is most likely the result of a rare mutation and is not a naturally occurring variation and other sites must account for the variance in virulence seen in natural isolates of CVB3.

Genetics of coxsackievirus B cardiovirulence and inflammatory heart muscle disease.

Coxsackieviruses B (CVBs) are etiological agents of human inflammatory myocardial disease. The genetics of the coxsackieviral virulence phenotype in mice are now beginning to be understood with the availability of infectious cDNA copies of CVB genomes. Investigations to date with CVB3 and CVB4 have shown that sites within a non-translated region and in the capsid proteins can affect the virulence phenotype. The relative importance of these sites to expression of the phenotype remains unclear.