Replication-competent or attenuated, nonpropagating vesicular stomatitis viruses expressing respiratory syncytial virus (RSV) antigens protect mice against RSV challenge.

Foreign glycoproteins expressed in recombinant vesicular stomatitis virus (VSV) can elicit specific and protective immunity in the mouse model. We have previously demonstrated the expression of respiratory syncytial virus (RSV) G (attachment) and F (fusion) glycoprotein genes in recombinant VSV. In this study, we demonstrate the expression of RSV F and G glycoproteins in attenuated, nonpropagating VSVs which lack the VSV G gene (VSVDeltaG) and the incorporation of these RSV proteins into recombinant virions. We also show that intranasal vaccination of mice with nondefective VSV recombinants expressing RSV G (VSV-RSV G) or RSV F (VSV-RSV F) elicited RSV-specific antibodies in serum (by enzyme-linked immunosorbent assay [ELISA]) as well as neutralizing antibodies to RSV and afford complete protection against RSV challenge. In contrast, VSVDeltaG-RSV F induced detectable serum antibodies to RSV by ELISA, but no detectable neutralizing antibodies, yet it still protected from RSV challenge. VSVDeltaG-RSV G failed to induce any detectable serum (by ELISA) or neutralizing antibodies and failed to protect from RSV challenge. The attenuated, nonpropagating VSVDeltaG-RSV F is a particularly attractive candidate for a live attenuated recombinant RSV vaccine.

Conservation of the respiratory syncytial virus SH gene.

Respiratory syncytial virus (RSV) encodes a short (64 or 65 amino acids) hydrophobic (SH) protein whose function in viral replication and pathogenesis is not understood. We carried out molecular epidemiological studies of the SH gene during the 1998-1999 seasonal epidemic in New Haven, Connecticut. Strains circulating during the epidemic were related to viruses identified worldwide. The SH gene transcriptional control signals were conserved in 70 (98.6%) of 71 isolates that we sequenced. The deduced amino acid sequence of the SH protein was nearly identical to subgroup A and subgroup B reference strains that were isolated in 1961 and 1962, respectively. Twenty-six (96.3%) of 27 subgroup A strains contained 0 or 1 amino acid substitution, compared with that of the reference A2 strain. Most subgroup B isolates (38 [86.4%] of 44 strains) contained 0, 1, or 2 amino acid substitutions, compared with that of the reference B18537 strain.

Respiratory syncytial virus vaccine development.

Respiratory syncytial virus is the major respiratory pathogen of infants and children worldwide. Currently, there is no effective vaccine to protect against respiratory syncytial virus infection. Immunoprophylaxis with hyperimmune globulin or with a humanized monoclonal antibody is expensive, limited to children with underlying disease, and not practical for general use. Antiviral therapy is controversial and of limited effectiveness. New approaches to the development of a vaccine for respiratory syncytial virus infection are promising. Several subunit vaccines and live attenuated virus vaccines are immunogenic and safe in children and adults. This review focuses on potential vaccine candidates and the challenges these candidate vaccines must overcome.

Fever without apparent source on clinical examination, lower respiratory infections in children, and enterovirus infections.

This section focuses on issues in infectious disease that are commonly encountered in pediatric office practice. McCarthy discusses recent literature regarding the evaluation and management of acute fevers without apparent source on clinical examination in infants and children and the evaluation of children with prolonged fevers of unknown origin. Klig reviews recent literature about lower respiratory tract infection in children. Finally, Kennedy and Kahn discuss recent developments in infectious diseases pertinent to office practice.

Fever without apparent source on clinical examination, lower respiratory infections in children, and other infectious diseases.

This section focuses on issues in infectious disease that are commonly encountered in pediatric office practice. Paul McCarthy discusses recent literature regarding the evaluation and management of acute fevers without apparent source on clinical examination in infants and children and the evaluation of children with prolonged fevers of unknown origin. Jean Klig reviews recent literature about lower respiratory tract infection in children. Finally, Jeffrey Kahn discusses recent developments concerning rotavirus vaccine.

Recombinant vesicular stomatitis virus expressing respiratory syncytial virus (RSV) glycoproteins: RSV fusion protein can mediate infection and cell fusion.

The genes encoding the respiratory syncytial virus (RSV) attachment (G) and fusion (F) envelope glycoproteins were expressed separately as additional genes in recombinant vesicular stomatitis viruses (VSV). Cells infected with the VSV-RSV F recombinant formed large syncytia illustrating the fusion activity of F in absence of other RSV proteins. Both F and G glycoproteins were expressed at the cell surface and incorporated into virions. Incorporation of these proteins did not require cytoplasmic tail sequences of VSV G. Using a compound, ammonium chloride, that raises the endosomal pH, we showed that presence of the RSV F glycoprotein in the envelope of recombinant VSV allowed for infectivity through a low-pH-independent pathway. Recombinant VSV expressing RSV glycoproteins could be useful as an RSV vaccine.

Fever without apparent source on clinical examination, infectious diseases, and lower respiratory infections in children.

This section focuses on issues in infectious diseases that are commonly encountered in pediatric office practice. Paul McCarthy discusses recent literature regarding the evaluation and management of acute fevers without apparent source on clinical examination in infants and children, and the evaluation of children with prolonged fevers of unknown origin. Jean Klig reviews recent literature about lower respiratory tract infection in children. Jeffrey Kahn and Eugene Shapiro discuss recent developments in pediatric infectious diseases concerning neonatal herpes infections, poliovirus immunization schedule, and group B streptococcus screening and treatment.

Fever without apparent source on clinical examination, lower respiratory infections in children, other infectious diseases, and acute gastroenteritis and diarrhea of infancy and early childhood.

This section focuses on issues in infectious disease that are commonly encountered in pediatric office practice. Paul McCarthy discusses recent literature regarding the evaluation and management of acute fevers without apparent source on clinical examination in infants and children and the evaluation of children with prolonged fevers of unknown origin. Jean Klig reviews recent literature about lower respiratory tract infection in children. Jeffrey Kahn and Eugene Shapiro discuss literature concerning several infectious diseases commonly seen in office settings and concerning which recent developments are of interest. Michael Baron reviews recent literature about gastroenteritis and diarrhea of infancy and early childhood.

A mutation of the nucleoside triphosphate phosphohydrolase I (NPH-I) gene confers sensitivity of vaccinia virus to interferon.

The role of vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH-I) in determining the resistance of this virus to alpha-interferon (IFN) was analyzed by using a well-defined temperature-sensitive mutant (ts36) in the NPH-I gene. Detailed analysis of viral proteins and mRNAs produced in cultured cells treated with or without IFN-alpha showed a strong inhibition by IFN of the synthesis of late virus polypeptides at the nonpermissive temperature, e.g., 83.8% inhibition for the luciferase reporter gene, which is associated with a decrease in steady-state mRNA levels. Sensitivity of ts36 to IFN was only the consequence of the point mutation in the NPH-1 gene, as shown by characterization of the rescued virus, R36. Our findings demonstrate that functional NPH-1 is required, at least in part, for resistance of vaccinia virus to IFN.

Effects of professional and media warnings about the association between aspirin use in children and Reye's syndrome.

The media played a central role in changing the use of aspirin among children with viral illness following reports of its association with a rare but deadly disease, Reye's syndrome (RS). It did so by alerting health professionals and parents about ways to prevent RS in children. Indeed, by the time aspirin product labeling was required by the FDA in 1986, most of the decline in RS incidence had already occurred. In the past, media-only health education campaigns have been relatively unsuccessful in achieving long-term changes in complex health behaviors. This article supports the theory that media warnings about the hazards of common products may successfully change consumer behaviors when the illness is devastating, the behavioral message is simple, acceptable and inexpensive alternatives are available, and the campaign is comprehensive, involving multiple professional groups to reinforce direct appeals to consumers.

Identification of the point mutations in two vaccinia virus nucleoside triphosphate phosphohydrolase I temperature-sensitive mutants and role of this DNA-dependent ATPase enzyme in virus gene expression.

The biological function of the nucleoside triphosphate phosphohydrolase I (NTPase I) enzyme of vaccinia virus is not yet known. In this investigation we have identified the genetic lesion of two temperature-sensitive mutants of vaccinia virus, ts50 and ts36, as single point mutations contained within the 5'615 nucleotides of the NTPase I gene (ts50, G to A at position 131; ts36, C to T at position 556). The point mutations result in amino acid substitutions of Gly to Glu-44 (ts50) and Pro to Ser-186 (ts36). In monkey BSC-40 cells, ts50 and ts36 behave phenotypically like wild-type virus with respect to replication and synthesis of viral DNA but are defective in late polypeptide synthesis. However, these two ts mutants displayed a drastically different phenotype in virus-infected human HeLa cells at the restrictive temperature; viral DNA replication did not occur and late polypeptide synthesis was absent. Moreover, if the early block was overcome by a temperature shift-up, then HeLa cells infected with the ts mutants displayed a profile characteristic of defective late viral polypeptide synthesis. Our results reveal that vaccinia NTPase I enzyme functions early and late in the viral replication cycle and that the phenotype of these ts mutants is dependent upon the cell type.

The cis-acting DNA sequences required in vivo for bacteriophage Mu helper-mediated transposition and packaging.

The 37,000 bp double-stranded DNA genome of bacteriophage Mu behaves as a plaque-forming transposable element of Escherichia coli. We have defined the cis-acting DNA sequences required in vivo for transposition and packaging of the viral genome by monitoring the transposition and maturation of Mu DNA-containing pSC101 and pBR322 plasmids with an induced helper Mu prophage to provide the trans-acting functions. We found that nucleotides 1 to 54 of the Mu left end define an essential domain for transposition, and that sequences between nucleotides 126 and 203, and between 203 and 1,699, define two auxiliary domains that stimulate transposition in vivo. At the right extremity, the essential sequences for transposition require not more than the first 62 base pairs (bp), although the presence of sequences between 63 and 117 bp from the right end increases the transposition frequency about 15-fold in our system. Finally, we have delineated the pac recognition site for DNA maturation to nucleotides 32 to 54 of the Mu left end which reside inside of the first transposase binding site (L1) located between nucleotides 1-30. Thus, the transposase binding site and packaging domains of bacteriophage Mu DNA can be separated into two well-defined regions which do not appear to overlap.

Molecular cloning, encoding sequence, and expression of vaccinia virus nucleic acid-dependent nucleoside triphosphatase gene.

A rabbit poxvirus genomic library contained within the expression vector lambda gt11 was screened with polyclonal antiserum prepared against vaccinia virus nucleic acid-dependent nucleoside triphosphatase (NTPase)-I enzyme. Five positive phage clones containing from 0.72- to 2.5-kilobase-pair (kbp) inserts expressed a beta-galactosidase fusion protein that was reactive by immunoblotting with the NTPase-I antibody. Hybridization analysis allowed the location of this gene within the vaccinia HindIIID restriction fragment. From the known nucleotide sequence of the 16-kbp vaccinia HindIIID fragment, we identified a region that contains a 1896-base open reading frame coding for a 631-amino acid protein. Analysis of the complete sequence revealed a highly basic protein, with hydrophilic COOH and NH2 termini, various hydrophobic domains, and no significant homology to other known proteins. Translational studies demonstrate that NTPase-I belongs to a late class of viral genes. This protein is highly conserved among Orthopoxviruses.

Characteristics of married and unmarried adolescent mothers and their partners.

PIP: In a middle class, urban clinic sample of 275 mostly Caucasian, adolescent mothers and their partners living in Utah, 3 groups were identified and their psychosocial characteristics were compared. Couples married at the time of conception (N=22) enjoyed more positive responses from prospective grandparents and earned more than couples not married at the time of conception. On the other hand, these initially married youths were much more likely to be high school dropouts, which suggests limits in their lifetime earning capacities, and they were not more likely to identify one another as sources of emotional support. Couples who married between conception and delivery (N=110) reported that prospective grandparents responded less favorably to news of the pregnancy than did relatives of the initially married couples, but while their current salaries were lower, they were much more likely to be continuing with their education. Those who married after conception also had fewer antisocial and conduct disorders than young men and women who chose to continue in a dating relationship (N=29). Overall, the couples who married after conception appeared to face less severe problems than either the initially married couples or the steady daters.^ieng

Orientation and sequence analysis of right ends and target sites of bacteriophage mu and D108 insertions in the plasmid pSC101.

We have isolated four independent insertions of the entire 37-kb D108cts 10 genome in the low-copy-number plasmid pSC101 in vivo. They were all formed by replicative transposition during the D108 lytic cycle. The orientation of these four insertions was found to be the same, with the left ends facing towards pSC101 replication, and the right end facing in the direction of all pSC101 transcription, as was previously found for a Mucts62 insertion in pSC101, pMC321. The exact sites of insertion of two of the D108 prophages, as well as the Mu prophage, have been determined by sequence analysis. All three insertions caused a 5-bp duplication of pSC101 sequences at the target site, as has been found for insertions formed by conservative integration upon lysogeny. Moreover, we have determined the nucleotide sequence of the first 75 bp of the right end of D108 and, though this end is interchangeable with the right end of Mu as a substrate for either phage's transposition functions, there are a number of nucleotide differences between them.

Activation of Coupling Factor 1 from Euglena gracilis Chloroplasts : Conditions for Optimal Activation and Their Possible Physiological Significance.

The recently described method for the activation of the Ca(2+)-ATPase of coupling factor 1 from chloroplasts (CF(1)) of Euglena gracilis by low pH occurs optimally in high concentrations of NaCl, and is unaffected by the acid used to lower the pH to 4.5. Activation is inhibited by light, and this effect can be reversed by the presence of NADP(+), ADP + inorganic phosphate, or an uncoupler. There appears to be no difference between the activities in the soluble and the particulate phases, and they seem to represent the same enzyme. The response of the activation process to light and to effectors of electron transport and phosphorylation indicates a possible physiological role for the acid activation of Euglena CF(1).

A Novel Method for the Activation and Solubilization of Coupling Factor 1 from Chloroplasts of Euglena gracilis.

A method was found for the in situ activation of the latent Ca(2+)-ATPase of the coupling factor from chloroplasts of Euglena gracilis and its resultant solubilization. The activation causes the concomitant solubilization of the enzyme and facilitates easy and nearly complete extraction. The activation consists of lowering the pH of isolated chloroplasts for 2 minutes to pH 4.5 to 4.7 with acetic acid, followed by neutralization. Increases in activity as high as 18-fold can be obtained. The method does not appear to work with chloroplasts from other sources.

The epsilon Subunit of the Chloroplast Coupling Factor 1 from Euglena gracilis: A Possible Role in Controlling ATPase Activity.

The coupling factor from chloroplasts (CF(1)) of Euglena gracilis Z strain is an active ATPase in situ, and its activity cannot be increased by treatment with trypsin or heating as is the case with the CF(1) from other sources. The smallest subunit of CF(1), the epsilon subunit, is supposed to be involved in controlling the ATPase activity. We have devised a simple technique for rapid and large-scale isolation of this subunit. The epsilon subunit from Euglena CF(1), although having only a limited inhibitory effect on Euglena CF(1), drastically inhibited the ATPase activity of heat-activated spinach CF(1). The inhibition of spinach CF(1) could be reversed by passage through Sephadex G-50 or by a second heat activation. An antibody to the epsilon subunit of Euglena CF(1) cross-reacted only weakly with CF(1) from spinach, Sorghum, Kalanchoë, or Anacystis nidulans, but reacted well with whole Euglena CF(1) in addition to its epsilon subunit. The antibody increased the ATPase activity of Euglena and Anacystis CF(1) and of unactivated or partially activated spinach CF(1). The results suggest that the function of the epsilon subunit in Euglena CF(1) is similar to its function in CF(1) from other sources. The data also suggest that changes induced in spinach CF(1) by activation involves modifications in subunits other than the epsilon one.