Targeting of Streptococcus mutans Biofilms by a Novel Small Molecule Prevents Dental Caries and Preserves the Oral Microbiome.

Dental caries is a costly and prevalent disease characterized by the demineralization of the tooth's enamel. Disease outcome is influenced by host factors, dietary intake, cariogenic bacteria, and other microbes. The cariogenic bacterial species Streptococcus mutans metabolizes sucrose to initiate biofilm formation on the tooth surface and consequently produces lactic acid to degrade the tooth's enamel. Persistence of S. mutans biofilms in the oral cavity can lead to tooth decay. To date, no anticaries therapies that specifically target S. mutans biofilms but do not disturb the overall oral microbiome are available. We screened a library of 2-aminoimidazole antibiofilm compounds with a biofilm dispersion assay and identified a small molecule that specifically targets S. mutans biofilms. At 5 µM, the small molecule annotated 3F1 dispersed 50% of the established S. mutans biofilm but did not disperse biofilms formed by the commensal species Streptococcus sanguinis or Streptococcus gordonii. 3F1 dispersed S. mutans biofilms independently of biofilm-related factors such as antigen I/II and glucosyltransferases. 3F1 treatment effectively prevented dental caries by controlling S. mutans in a rat caries model without perturbing the oral microbiota. Our study demonstrates that selective targeting of S. mutans biofilms by 3F1 was able to effectively reduce dental caries in vivo without affecting the overall oral microbiota shaped by the intake of dietary sugars, suggesting that the pathogenic biofilm-specific treatment is a viable strategy for disease prevention.

Fecal metabolomics in pediatric spondyloarthritis implicate decreased metabolic diversity and altered tryptophan metabolism as pathogenic factors.

We have previously shown alterations in the composition of the gut microbiota in children with enthesitis-related arthritis (ERA). To explore the mechanisms by which an altered microbiota might predispose to arthritis, we performed metabolomic profiling of fecal samples of children with ERA. Fecal samples were collected from two cohorts of children with ERA and healthy control subjects. Nano-liquid chromatography-mass spectroscopy (LC-MS) was performed on the fecal water homogenates with identification based upon mass: charge ratios. Sequencing of the 16S ribosomal DNA (rDNA) on the same stool specimens was performed. In both sets of subjects, patients demonstrated lower diversity of ions and under-representation of multiple metabolic pathways, including the tryptophan metabolism pathway. For example, in the first cohort, out of 1500 negatively charged ions, 154 were lower in ERA patients, compared with only one that was higher. Imputed functional annotation of the 16S ribosomal DNA sequence data demonstrated significantly fewer microbial genes associated with metabolic processes in the patients compared with the controls (77 million versus 58 million, P=0.050). Diminished metabolic diversity and alterations in the tryptophan metabolism pathway may be a feature of ERA.

Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2015).

Changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses in February 2015 are listed.

Tracking the changes in virus taxonomy.

A database and website ( http://www.ictvonline.org/taxonomyReleases.asp ) have been established where the history of changes in virus taxonomy from 1971 to the present day can easily be traced. Each change is linked to a source document confirming the change or, for most changes since 2002, to the taxonomic proposal approved by the International Committee on Taxonomy of Viruses (ICTV).

Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2014).

Changes to virus taxonomy approved by a vote of all ICTV members in February-March 2014 are reported.

A phylogenetic analysis using full-length viral genomes of South American dengue serotype 3 in consecutive Venezuelan outbreaks reveals a novel NS5 mutation.

Dengue virus currently causes 50-100 million infections annually. Comprehensive knowledge about the evolution of Dengue in response to selection pressure is currently unavailable, but would greatly enhance vaccine design efforts. In the current study, we sequenced 187 new dengue virus serotype 3 (DENV-3) genotype III whole genomes isolated from Asia and the Americas. We analyzed them together with previously-sequenced isolates to gain a more detailed understanding of the evolutionary adaptations existing in this prevalent American serotype. In order to analyze the phylogenetic dynamics of DENV-3 during outbreak periods; we incorporated datasets of 48 and 11 sequences spanning two major outbreaks in Venezuela during 2001 and 2007-2008, respectively. Our phylogenetic analysis of newly sequenced viruses shows that subsets of genomes cluster primarily by geographic location, and secondarily by time of virus isolation. DENV-3 genotype III sequences from Asia are significantly divergent from those from the Americas due to their geographical separation and subsequent speciation. We measured amino acid variation for the E protein by calculating the Shannon entropy at each position between Asian and American genomes. We found a cluster of seven amino acid substitutions having high variability within E protein domain III, which has previously been implicated in serotype-specific neutralization escape mutants. No novel mutations were found in the E protein of sequences isolated during either Venezuelan outbreak. Shannon entropy analysis of the NS5 polymerase mature protein revealed that a G374E mutation, in a region that contributes to interferon resistance in other flaviviruses by interfering with JAK-STAT signaling was present in both the Asian and American sequences from the 2007-2008 Venezuelan outbreak, but was absent in the sequences from the 2001 Venezuelan outbreak. In addition to E, several NS5 amino acid changes were unique to the 2007-2008 epidemic in Venezuela and may give additional insight into the adaptive response of DENV-3 at the population level.

Evidence for separation of HCV subtype 1a into two distinct clades.

The nucleotide sequence diversity present among hepatitis C virus (HCV) isolates allows rapid adjustment to exterior forces including host immunity and drug therapy. This viral response reflects a combination of a high rate of replication together with an error-prone RNA-dependent RNA polymerase, providing for the selection and proliferation of the viruses with the highest fitness. We examined HCV subtype 1a whole-genome sequences to identify positions contributing to genotypic and phenotypic diversity. Phylogenetic tree reconstructions showed two distinct clades existing within the 1a subtype with each clade having a star-like tree topology and lacking definite correlation between time or place of isolation and phylogeny. Identification of significant phylogenetically informative sites at the nucleotide level revealed positions not only contributing to clade differentiation, but which are located at or proximal to codons associated with resistance to protease inhibitors (NS3 Q41) or polymerase inhibitors (NS5B S368). Synonymous/nonsynonymous substitution mutation analyses revealed that the majority of nucleotide mutations yielded synonymous amino acids, indicating the presence of purifying selection pressure across the polyprotein with pockets of positive selection also being detected. Despite evidence for divergence at several loci, certain 1a characteristics were preserved including the length of the alternative reading frame/F protein (ARF/F) gene, and a subtype 1a-specific phosphorylation site in NS5A (S349). Our analysis suggests that there may be strain-specific differences in the development of antiviral resistance to viruses infecting patients who are dependent on the genetic variation separating these two clades.

Poxviruses: past, present and future.

The analysis of poxvirus genomes is complex, in part, because of their size (130-360 kb) and the fact that gene content is variable; a common set of 49 genes has been found in all sequenced poxviruses and an additional 41 genes are also present in all sequenced orthopoxviruses. As a group, poxviruses have a very broad range of eukaryotic hosts (including mammals, birds, reptiles and insects) and many poxvirus genes are associated with blocking host anti-viral responses. One consequence of this is that many poxvirus genes are not essential for growth in tissue culture and that extensive passaging in vitro results in the accumulation of mutations, including deletions that result in loss of gene function. Here, we review various comparative analyses of the poxviruses including gene prediction, gene conservation and function, genome organization, and poxvirus taxonomy and evolution.

Complete coding sequences of the rabbitpox virus genome.

Rabbitpox virus (RPXV) is highly virulent for rabbits and it has long been suspected to be a close relative of vaccinia virus. To explore these questions, the complete coding region of the rabbitpox virus genome was sequenced to permit comparison with sequenced strains of vaccinia virus and other orthopoxviruses. The genome of RPXV strain Utrecht (RPXV-UTR) is 197 731 nucleotides long, excluding the terminal hairpin structures at each end of the genome. The RPXV-UTR genome has 66.5 % A + T content, 184 putative functional genes and 12 fragmented ORF regions that are intact in other orthopoxviruses. The sequence of the RPXV-UTR genome reveals that two RPXV-UTR genes have orthologues in variola virus (VARV; the causative agent of smallpox), but not in vaccinia virus (VACV) strains. These genes are a zinc RING finger protein gene (RPXV-UTR-008) and an ankyrin repeat family protein gene (RPXV-UTR-180). A third gene, encoding a chemokine-binding protein (RPXV-UTR-001/184), is complete in VARV but functional only in some VACV strains. Examination of the evolutionary relationship between RPXV and other orthopoxviruses was carried out using the central 143 kb DNA sequence conserved among all completely sequenced orthopoxviruses and also the protein sequences of 49 gene products present in all completely sequenced chordopoxviruses. The results of these analyses both confirm that RPXV-UTR is most closely related to VACV and suggest that RPXV has not evolved directly from any of the sequenced VACV strains, since RPXV contains a 719 bp region not previously identified in any VACV.

Genome of the bacterium Streptococcus pneumoniae strain R6.

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

The complete sequence of the mucosal pathogen Ureaplasma urealyticum.

The comparison of the genomes of two very closely related human mucosal pathogens, Mycoplasma genitalium and Mycoplasma pneumoniae, has helped define the essential functions of a self-replicating minimal cell, as well as what constitutes a mycoplasma. Here we report the complete sequence of a more distant phylogenetic relative of those bacteria, Ureaplasma urealyticum (parvum biovar), which is also a mucosal pathogen of humans. It is the third mycoplasma to be sequenced, and has the smallest sequenced prokaryotic genome except for M. genitalium. Although the U. urealyticum genome is similar to the two sequenced mycoplasma genomes, features make this organism unique among mycoplasmas and all bacteria. Almost all ATP synthesis is the result of urea hydrolysis, which generates an energy-producing electrochemical gradient. Some highly conserved eubacterial enzymes appear not to be encoded by U. urealyticum, including the cell-division protein FtsZ, chaperonins GroES and GroEL, and ribonucleoside-diphosphate reductase. U. urealyticum has six closely related iron transporters, which apparently arose through gene duplication, suggesting that it has a kind of respiration system not present in other small genome bacteria The genome is only 25.5% G+C in nucleotide content, and the G+C content of individual genes may predict how essential those genes are to ureaplasma survival.

Identification of the human pituitary tumor transforming gene (hPTTG) family: molecular structure, expression, and chromosomal localization.

In an attempt to determine the mechanism of human tumorigenesis, we have searched for oncogenes and recently reported the molecular cloning of a potent oncogene (hPTTG) from human testis. hPTTG mRNA is expressed at high levels in various human tumors and tumor cell lines. Overexpression of hPTTG in the mouse fibroblast cell line (NIH 3T3) results in an increase in cell proliferation, induces cellular transformation in vitro, and promotes tumor formation in nude mice. The hPTTG gene isolated from the human genomic library consists of five exons and four introns and spans over 10kb. In the studies reported here, we further investigated the possibility of the presence of additional genes homologous to hPTTG in the human genome, which was first indicated by Southern blot analysis of the human genomic DNA and chromosomal mapping of the hPTTG gene using DNA from humanxhamster hybrid cell lines in PCR. Sequencing and restriction map analysis of the additional genomic clones identified two intronless genes homologous to hPTTG. This finding was confirmed by the chromosomal location of the second gene to chromosome 4p15.1 and the third gene to chromosome 8q13.1. Based on the similarity in sequences, we proposed that hPTTG be renamed hPTTG1 and the new genes be named hPTTG2 and hPTTG3. hPPTG2 was found to be 91% identical and hPPTG3 89% identical with hPPTG1 at the amino acid level. Northern blot and reverse transcriptase/polymerase chain reaction (RT/PCR) analyses of the mRNA from various human tissues revealed differential expression of the hPTTG2 and hPTTG3 genes in normal and tumor tissues, suggesting that these genes may be associated with tumorigenesis.

The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association.

The E2 protein of papillomaviruses is a site-specific DNA binding nuclear protein. It functions as the primary replication origin recognition protein and assists in the assembly of the preinitiation complex. It also helps regulate transcription from the native viral promoter. The E2 protein consists of an amino-terminal (N) trans-acting domain, a central hinge (H) domain, and a carboxyl-terminal (C) protein dimerization and DNA binding domain. The hinge is highly divergent among papillomaviruses, and little is known about its functions. We fused the enhanced green fluorescent protein (GFP) with the full-length human papillomavirus type 11 (HPV-11) E2 protein and showed that the resultant fusion, called gfpE2, maintained transcription and replication functions of the wild-type protein and formed similar subnuclear foci. Using a series of GFP fusion proteins, we showed that the hinge conferred strong nuclear localization, whereas the N or C domain was present in both cytoplasm and nucleus. Biochemical fractionation demonstrated that the N domain and hinge, but not the C domain, independently associated with the nuclear matrix. Mutational analyses showed that a cluster of basic amino acid residues, which is conserved among many mucosotropic papillomaviruses, was required for efficient nuclear localization and nuclear matrix association. This mutation no longer repressed the HPV-11 upstream regulatory region-controlled reporter expression. However, a very small fraction of this mutant colocalized with E1 in the nucleus, perhaps by a piggyback mechanism, and was able to support transient replication. We propose that the hinge is critical for the diverse regulatory functions of the HPV-11 E2 protein during mRNA transcription and viral DNA replication.

Genetic variability among group A and group B respiratory syncytial viruses in a children's hospital.

Respiratory syncytial (RS) viruses isolated over three epidemic periods in a children's hospital in the United States were analyzed. The viruses (n = 174) were characterized as to major antigenic group (group A or B) by a PCR-based assay. Group A RS viruses were dominant the first 2 years, followed by a year with group B dominance (ratios of group A to group B viruses for epidemic periods, 56/4 for 1993-1994, 42/3 for 1994-1995, and 19/50 for 1995-1996). Genetic variability within the groups was assessed by restriction fragment analysis of PCR products; 79 isolates were also analyzed by nucleotide sequence determination of a variable region of the glycoprotein G gene. Among the group A RS virus isolates, this G-protein variable region had amino acid differences of as great as 38%. The G-protein amino acids of the group A viruses differed by up to 31% from the G-protein amino acids of a prototype (A2) group A virus. Among the group B RS virus G proteins, amino acid differences were as great as 14%. The G-protein amino acids of the group B viruses differed by up to 27% from the G-protein amino acids of a prototype (18537) group B virus. The group A and group B RS viruses demonstrated genetic variability between years and within individual years. Phylogenetic analysis revealed that there were multiple evolutionary lineages among both the group A and group B viruses. Among the recent group B isolates, variability was less than that seen for the group A viruses. However, comparisons to prototype strains revealed that the group B RS viruses may vary more extensively than was observed over the 3 years studied in the present investigation.

Phylogenetic analysis of the 16S-23S rRNA intergenic spacer regions of the genus Ureaplasma.

The nucleotide sequences of the spacer regions between the 16S and 23S rRNA genes of six Ureaplasma species were determined following amplification by polymerase chain reaction. The hypothetical secondary structure of the spacer regions was divided into four domains. Domains I and III were highly conserved among different Ureaplasma species but domains II and IV were variable. The intergenic spacer regions of ureaplasmas lacked spacer tRNA genes. Phylogenetic relationships among the Ureaplasma species indicated that the evolutionary history of the Ureaplasma species follows that of the host animal species, suggesting coevolution between ureaplasmas and animals.

Complementation of a vesicular stomatitis virus glycoprotein G mutant with wild-type protein expressed from either a bovine papilloma virus or a vaccinia virus vector system.

Using a complementation assay, we have evaluated the potential of two eukaryotic expression systems to produce functional virus proteins. The first expression system was based on a bovine papilloma virus (BPV) eukaryotic expression vector which contained a copy of the gene for the membrane glycoprotein G of vesicular stomatitis virus (VSV). This vector was transfected into a mouse cell line, and transformed cell clones constitutively expressing VSV G protein were selected. These cell clones were then screened for their ability to support the replication of a temperature-sensitive G mutant of VSV (tsO45) at the permissive and nonpermissive temperatures. A 100-fold increase in tsO45 titer was observed in some of the G protein-producing cell lines in comparison with nonproducing cells. These results were compared with complementation by VSV G protein expressed from a second expression system utilizing a vaccinia virus (VV) recombinant which produced bacteriophage T7 RNA polymerase. T7 RNA polymerase expressed in cells infected with the vaccinia recombinant produced VSV G transcripts from a plasmid which had been transfected into these cells. This plasmid contained the VSV G gene cloned between T7 RNA polymerase initiation and termination signals. VSV G protein expressed by this system was able to complement tsO45 replication at the nonpermissive temperature, and yielded much greater levels of complemented virus than the BPV system. When calcium phosphate-mediated transfection was used to introduce the VSV G plasmid vector into cells infected with the VV recombinant, a complementation efficiency as high as 1500-fold was obtained. Using lipofectin-mediated transfection, a 15,000-fold increase in virus titer could be obtained in G protein-producing cells in contrast to nonproducing cells. At the nonpermissive temperature, yields of temperature-sensitive virus were within 10-fold of the yields obtained at the permissive temperature. Virus produced in this system was shown to be a pseudotype which contained wild-type G protein in the viral envelope but still maintained the temperature-sensitive genotype. This expression system will be used to study the extent to which the integrity of the G coding sequence of wild-type VSV might be altered in the absence of selection pressure for functional G protein during VSV replication.

An inhibitor of interferon action: II. Biological properties of the IFN-gamma-associated inhibitor of interferon action.

Previously an inhibitor of interferon action had been isolated from mitogen stimulated mouse spleen cells. This inhibitor was associated with the IFN-gamma molecule and might possibly represent an altered IFN-gamma molecule which can no longer induce an effective antiviral state. This report further investigates the biological properties of this inhibitor. Inhibitor activity is independent of the virus used in the interferon assay. Inhibitor activity has also been found to be species specific. Mouse inhibitor does not inhibit the antiviral activity of either human IFN-gamma or human IFN-beta. However, inhibitor production is not limited to the mouse system. Inhibitor is also present in preparations of human IFN-gamma. The inhibitor does not appear to directly compete with IFN-gamma for a specific cell surface receptor since inhibitor activity is independent of interferon concentration. The presence of inhibitor allows a unique, albeit reduced level of antiviral protection to develop. Increasing concentrations of interferon do not increase the level of antiviral protection allowed by the inhibitor. This inhibitor of interferon action may represent a natural mechanism whereby IFN-gamma-induced effects are regulated in vivo.

An inhibitor of interferon action: I. Physical association of the inhibitor with interferon-gamma.

Previously, an inhibitor of interferon action has been identified in mouse lymphokine preparations. The inhibitor was produced following IFN-gamma production and thus may be involved in regulation of IFN-gamma action. We now show that native inhibitor activity as isolated from crude and partially purified IFN-gamma preparations migrates along with interferon activity on molecular sieving gels. IFN-gamma which comigrates with inhibitor migrates at a slightly higher molecular weight than IFN-gamma free of inhibitor. This inhibitor activity correlates with the decreased antiviral activity of crude IFN-gamma produced late in the course of mitogen stimulation. This is apparent for both plaque reduction and yield reduction assays for interferon. This association of IFN-gamma with its inhibitor suggests a mechanism for the local regulation of IFN-gamma activity.