Molecular epidemiology and genetic diversity of echovirus type 30 (E30): genotypes correlate with temporal dynamics of E30 isolation.

Echovirus type 30 (E30) (genus, Enterovirus; family, Picornaviridae) has caused large outbreaks of aseptic meningitis in many regions of the world in the last 40 years. U.S. enterovirus surveillance data for the period 1961 to 1998 indicated that the annual proportion of E30 isolations relative to total enterovirus isolations has fluctuated widely, from a low of 0% in 1966 to a high of 42% in 1998. Peaks of E30 isolations occurred in the years 1968 to 1969, 1981 to 1984, 1990 to 1993, and 1997 to 1998, coincident with large nationwide outbreaks of E30-associated aseptic meningitis. Analysis of the complete VP1 sequence (876 nucleotides) of 136 E30 strains isolated in geographically dispersed regions of the United States and nine other countries between 1956 and 1998 indicated that the currently circulating E30 strains are genetically distinct from those isolated 30 to 40 years ago. Phylogenetic reconstruction demonstrated the existence of at least four distinct genetic groups, three of which have not been isolated in North America since 1981. Two of the three groups disappeared during periods when E30 was isolated infrequently. All North American E30 strains isolated after 1988 were closely related to one another, and all post-1993 isolates were of the same lineage within this group. Surveillance data indicate that E30 causes large national outbreaks of 2- to 4-year durations, separated by periods of relative quiescence. Our results show that shifts in the overall genetic diversity of E30 and the predominant genetic type correlate temporally with the dynamics of E30 isolation. The sequence data also provide a basis for the application of molecular techniques for future epidemiologic investigations of E30 disease.

New syndrome of spondylospinal thoracic dysostosis with multiple pterygia and arthrogryposis.

We describe a "new" syndrome of spondylospinal thoracic dysostosis with a short curved spine and fusion of the spinous processes, short thorax with "crab-like" configuration of the ribs, pulmonary hypoplasia, severe arthrogryposis and multiple pterygia, and hypoplastic maxilla and mandible in two siblings. This appears to be an autosomal recessive lethal trait. A literature review revealed two reports of four similar or related cases.

An etoposide-induced block in vaccinia virus telomere resolution is dependent on the virus-encoded DNA ligase.

Etoposide, an inhibitor of the breakage-reunion reaction associated with cellular type II DNA topoisomerases, was shown to inhibit plaque formation of vaccinia virus. This drug had a major effect on the segregation of newly replicated DNA concatemers. Gene expression and the initiation and elongation phases of viral DNA replication were essentially unaffected. Pulsed-field gel electrophoresis of viral DNA replicated in the presence of etoposide revealed two major classes of DNA: the mature monomeric linear genome and DNA that failed to enter the gel (the relative proportions depending on the concentrations of drug). Restriction enzyme analysis showed a severe defect in telomere resolution. In addition, slowly migrating restriction fragments were suggestive of a general recombination defect. We have isolated several etoposide-resistant mutants and used marker rescue and DNA sequencing to localize the resistance-causing mutation to the amino terminus of the viral DNA ligase gene. Inactivation of the DNA ligase also resulted in an etoposide-resistant phenotype, but to a lesser extent. The telomere resolution and segregation defects were corrected both in the drug-resistant mutants and in the DNA ligase knockout mutants. Reinsertion of wild-type or mutant DNA ligase in the viral thymidine kinase locus confirmed the role of the viral DNA ligase in conferring sensitivity not only to etoposide but also to another topoisomerase II inhibitor, 4'-(9-acridinylamino) methanesulphon-m-anisidide (mAMSA). The data suggest that the nonessential DNA ligase is involved in telomere resolution, possibly as part of a general recombinase.

The vaccinia virus-encoded uracil DNA glycosylase has an essential role in viral DNA replication.

The vaccinia virus conditional-lethal temperature-sensitive (ts) mutant ts4149 is, at the nonpermissive temperature, severely impaired in its ability to replicate its DNA genome. Compared to wild type, the amount of replication is suppressed by several orders of magnitude, and the little DNA that is replicated is not converted to mature linear genomes. We have demonstrated that this "DNA-" phenotype is not the result of a failure to produce early proteins. In agreement with the DNA- phenotype, intermediate and late gene expression were not detected. Marker rescue and DNA sequencing located the mutation in ts4149 to open reading frame D4. This gene has recently been shown to encode a 25-kDa protein with uracil DNA glycosylase activity (D. T. Stuart, C. Upton, M. A. Higman, E. G. Niles, and G. McFadden (1993), J. Virol. 67, 2503-2512). We speculate on the function of this "essential" viral repair enzyme and its role(s) in viral DNA replication.

Identification of a temperature-sensitive mutant of vaccinia virus defective in late but not intermediate gene expression.

The vaccinia virus conditional-lethal temperature-sensitive (ts) mutant tsC63 is defective in the synthesis of some but not all postreplicative proteins. Synthesis of the temporal "intermediate" class of proteins was unaffected, whereas "late" proteins were absent at the nonpermissive temperature. At the DNA level, DNA synthesis was unaffected, but telomere resolution was severely inhibited. In order to identify the defective gene responsible for this ts defect, we performed marker rescue and DNA sequencing experiments. We localized the lesion to open reading frame (ORF) A1L, which has recently been identified as one of the three intermediate genes required for the transcription of late genes (J.G. Keck, C.J. Baldick, Jr., and B. Moss, (1990). Cell 61, 801-809). S1 nuclease analysis of viral mRNA demonstrated that the ts defect in late protein synthesis was caused by a defect in the transcription of stable mRNA and therefore provides evidence for a role of the A1L gene product during in vivo transcriptional activation of late genes or stabilization of late RNA. Furthermore, the kinetics of early protein synthesis in tsC63-infected cells suggests that, in addition to its role in trans-activation of late genes, intermediate gene expression mediates suppression of early protein synthesis. The telomere resolution defect of this mutant is presumably a secondary consequence of the defect in late gene expression.

A temperature-sensitive lesion in the small subunit of the vaccinia virus-encoded mRNA capping enzyme causes a defect in viral telomere resolution.

Using pulsed-field gel electrophoresis, we demonstrated that the temperature-sensitive (ts) conditional lethal mutant ts9383 is, at the nonpermissive temperature, defective in the resolution of concatemeric replicative intermediate DNA to linear 185-kb monomeric DNA genomes. The resolution defect was shown to be the result of a partial failure of the mutant virus to convert the replicated form of the viral telomere to hairpin termini. In contrast to other mutants of this phenotype, pulse-labeling of viral proteins at various times postinfection revealed no obvious difference in the quantity or temporal appearance of members of the late class of polypeptides. Using the marker rescue technique, we localized the ts lesion in ts9383 to an approximately 1-kb region within the HindIII D fragment. Both the ts phenotype and the resolution defect were shown to be caused by a single-base C----T point mutation resulting in the conversion of the amino acid proline to serine in codon 23 of open reading frame D12. This gene encodes a 33-kDa polypeptide which is known to be the small subunit of the virus-encoded mRNA capping enzyme (E. G. Niles, G. J. Lee-Chen, S. Shuman, B. Moss, and S. S. Broyles, Virology 172:513-522, 1989). The data are consistent with a role for this capping enzyme subunit during poxviral telomere resolution.

Interrupted aortic arch. A conservative approach for the sick neonate.

Interrupted aortic arch with associated ventricular septal defect is a congenital cardiovascular defect which, untreated, is lethal in nearly 100% of the cases. We have treated nine patients by reconstructing the aorta with endogenous arch vessels; in five of them, concomitant pulmonary artery banding was also done. If two infants with preoperative complete renal failure are excluded, the mortality with this approach is only 29%. Long-term follow-up of these patients demonstrates excellent hemodynamic results with marked reduction of the anastomotic gradient in the older survivors. Growth of the anastomosis has been noted in the older survivors.