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.

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.

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.

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.

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.

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.

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.

Coxsackievirus B3: primary structure of the 5' non-coding and capsid protein-coding regions of the genome.

The nucleotide sequence from the 5' terminus to nucleotide 3822 has been determined for the genome of the enterovirus, coxsackievirus B3 (CB3). This region encompasses the 5'-terminal 738 nucleotide non-coding sequence and the region which codes for the four viral capsid proteins and for the initial products of the P2 region. Regions in the 5' non-translated RNA sequence which may be involved with a structural and/or regulatory role are discussed.

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.

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.

Can recombinant DNA technology provide useful vaccines against viruses which induce heart disease?

In the panoply of armaments against viral infections, both drugs and vaccines have been employed. Numerous vaccines have enjoyed spectacular success in either eradicating or controlling various viral diseases, whereas there are still few, effective anti-viral drugs. Coxsackie B viruses are agents of human inflammatory heart disease and may trigger events leading to a failing heart. We believe that enteroviral heart disease could be controlled or eradicated through the use of vaccines, in much the same manner as poliovirus-induced poliomyelitis has been controlled through vaccination. We present here preliminary data which deal with an approach to the development of enterovirus vaccines and the use of a chimeric coxsackievirus B3 (CVB3) vaccine in a murine model of CVB3-induced inflammatory heart disease.

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

A 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%.

Molecular cloning and partial characterization of the coxsackievirus B3 genome. Brief report.

The RNA genome of coxsackievirus B3 has been cloned and partially characterized by restriction mapping, partial sequence analysis, and hybridization to heterologous coxsackievirus B genomes. It differs significantly from the poliovirus genomic structure.

Reduced congenital cytomegalovirus (CMV) infection after maternal immunization with a guinea pig CMV glycoprotein before gestational primary CMV infection in the guinea pig model.

This study evaluated the effects of subunit guinea pig (GP) cytomegalovirus (CMV) immunization on congenital infection. Two 25-micrograms doses of an abundant GPCMV glycoprotein, the gp60-90 complex, plus adjuvant to GPs before pregnancy produced virus-specific humoral (neutralizing and ELISA) and cellular (proliferative and delayed type hypersensitivity) responses. Viral challenge before midgestation resulted in shorter maternal viremia in immunized than in unimmunized dams (3 vs. 17 days). Litters of immunized dams had reduced organ involvement and rates of congenital infection (48% vs. 18%) and increased mean birth weights (74 vs. 99 g). Amplification of DNA extracted from pup blood or organs with primers from the gB gene of GPCMV revealed congenital infection in some pups without detectable CMV by classic culture techniques. These data suggest that induction of preconception immunity to CMV by vaccine could be useful in reducing both the incidence and severity of congenital CMV disease.

Detection of enteroviruses in cell cultures by using in situ transcription.

In situ transcription (IST) was shown to be useful for the detection of human enteroviral RNA in cultured cells. A primer to detect a wide variety of enteroviral genomes and a coxsackievirus type B3 genome-specific primer were demonstrated to be efficient in IST assays. Transcription times greater than 10 to 30 min did not significantly improve the acquisition of a specific signal, whereas the signal-to-noise ratio decreased with time. Inclusion of actinomycin D to suppress DNA-dependent DNA polymerase activity in reverse transcriptase decreased the signal that was obtained without improving the signal-to-noise ratio. Use of RNase H-free murine leukemia virus reverse transcriptase in the IST reaction increased the signal versus that obtained by use of the avian myeloblastosis virus enzyme, which contains endogenous RNase H activity. Exogenous RNase H added to the transcription reaction ablated the signal. Background transcription because of poorly hybridized (mismatched) primers was reduced after primer hybridization and prior to the transcription reaction by rinsing fixed cells with 3 M tetramethylammonium chloride at temperatures which dissociate mismatched primer-template duplexes. The rapid detection time and the simplicity of application suggest that IST can be performed with a high specificity for the detection of enteroviral genomic sequences in cultured cells and may be more useful than in situ hybridization for the detection of enteroviral genomes.