RFI-641 inhibits entry of respiratory syncytial virus via interactions with fusion protein.

BACKGROUND: RFI-641, a small dendrimer-like compound, is a potent and selective inhibitor of respiratory syncytial virus (RSV), which is currently a clinical candidate for the treatment of upper and lower respiratory tract infections caused by RSV. RFI-641 inhibits RSV growth with an IC(50) value of 50 nM and prevents syncytia formation in tissue culture. RSV contains of three surface glycoproteins, a small hydrophobic (SH) protein of unknown function, and attachment (G) and fusion (F) proteins that enable binding and fusion of virus, respectively, with target cells. Because of their role in attachment and fusion, the G and F surface proteins are prominent targets for therapeutic intervention. RFI-641 was previously shown to bind purified preparations of RSV fusion protein. Based on this observation, in conjunction with the biological results, it was speculated that the fusion event might be the target of these inhibitors. RESULTS: A fusion assay based upon the relief of self-quenching of octadecyl rhodamine R18 was used to determine effects of the inhibitors on binding and fusion of RSV. The results show that RFI-641 inhibits both RSV-cell binding and fusion events. The inhibition of RSV is mediated via binding to the fusion protein on the viral surface. A closely related analog, WAY-158830, which is much less active in the virus-infectivity assay does not inhibit binding and fusion of RSV with Vero cells. CONCLUSIONS: RFI-641, an in vivo active RSV inhibitor, is shown to inhibit both binding and fusion of RSV with cells, events that are early committed steps in RSV entry and pathogenicity. The results described here demonstrate that a non-peptidic, small molecule can inhibit binding and fusion of enveloped virus specifically via interaction with the viral fusion protein.

Novel anti-RSV dianionic dendrimer-like compounds: design, synthesis and biological evaluation.

Human Respiratory Syncytial Virus (RSV) is considered to be the leading cause of lower respiratory tract disease in infants and young children. RSV is also a common pathogen in immunocompromised adults and in the elderly. RSV infection can be epidemic and is evident worldwide. Ribavirin, a small molecule agent, and Synagis, a monoclonal neutralizing antibody, are the only approved drugs for treatment and prevention of RSV in high-risk patients. This review is focused on a group of novel and specific inhibitors discovered at Wyeth-Ayerst Research. Some of these inhibitors have IC50 <50 nM and are active against all the tested group A and B viruses. They also have shown good efficacy in cotton rats and primates. Mechanism of action studies indicate that the compounds inhibit the next step in infection after adsorption suggesting that fusion is the target. A strong relationship between the inhibitor structures and their anti-RSV activity was established. This relationship appears to derive from a multivalent interaction between the functional groupings of the inhibitors and the F protein, which seem to be highly complementary and directional.

Two geographically distinct isolates of Borrelia burgdorferi from the United States share a common unique ancestor.

The genetic diversity of Borrelia burgdorferi isolates from several geographic regions was evaluated by nucleotide sequence analysis of the genes encoding 23S ribosomal RNA and outer surface protein A. Comparison of nucleotide sequences spanning 738 bp of the 23S ribosomal DNA from two unusual isolates, DN127 (Del Norte County, California) and 25015 (Millbrook, New York), to homologous sequences from other B. burgdorferi isolates from the United States and Russia identified several nucleotide sequence polymorphisms that are unique to these two isolates. Sequence analysis of a 615 nucleotide segment of the gene encoding outer surface protein A also revealed greater similarity of strains DN127 and 25015 (94.1%) compared to other US and Eurasian isolates. These data were further corroborated by genomic macrorestriction analysis, in which DN127 and 25015 demonstrated unique restriction digestion patterns. Our findings suggest that substantial genetic diversity of B. burgdorferi, rivaling that of European strains, exists among isolates from the United States. Strains DN127 and 25015 are unique among all B. burgdorferi isolates tested to date, and though isolated from opposite longitudinal extremes of the North American continent, are closely related.

Molecular typing of Borrelia burgdorferi sensu lato by PCR-restriction fragment length polymorphism analysis.

The etiologic agent of Lyme borreliosis, Borrelia burgdorferi sensu lato, has been isolated from many biologic sources in North America and Eurasia, and isolates have been divided into three distinct genospecies (B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii). In order to explore the possible association of genospecies with disease manifestation, 60 isolates of B. burgdorferi sensu lato were subjected to 5S rDNA-linked restriction fragment length polymorphism (RFLP) analysis. The results confirmed earlier studies which indicated that virtually all North American isolates are B. burgdorferi sensu stricto, whereas Eurasian strains fall into all three genospecies. Thirty-five isolates were further characterized by PCR amplification of a region of the 16S-23S rDNA spacer and HinfI digestion of the products. This method resulted in the subdivision of B. burgdorferi sensu stricto into two distinct PCR-RFLP types. In contrast, B. garinii isolates all displayed an identical pattern. Additionally, a number of previously unclassified North American isolates (25015, DN127, 19857, 24330) showed distinctively different PCR-RFLP patterns. The application of this method for the typing of uncultured B. burgdorferi directly in biologic samples was demonstrated by analysis of several field-collected Ixodes scapularis tick specimens. The described PCR-RFLP technique should allow for the direct and rapid molecular typing of B. burgdorferi-containing samples and facilitate studies of the relationship between spirochete genotype and clinical disease.

Sequence analysis of the ribosomal RNA operon of the Lyme disease spirochete, Borrelia burgdorferi.

An 11,955-bp region of the Borrelia burgdorferi chromosome containing all the genes encoding ribosomal RNA (rRNA) has been sequenced. The region contains a single gene encoding 16S rRNA and two genes encoding the 23S and 5S rRNAs. The sizes of the 16S, 23S and 5S rRNAs encoded by these genes are 1537, 2926 and 112 nucleotides, respectively. In addition, the genes encoding tRNA(Ala) and tRNA(Ile) are located in the intergenic spacer between the 16S and 23S rDNAs. The tDNAs do not encode the common CCA 3' end which presumably must be added posttranscriptionally. All the genes are present in the same orientation, except for that encoding tRNA(Ile), which is transcribed from the opposite strand. The latter implies that the rDNAs are not transcribed as a single unit. The location of putative promoters and termination signals in the sequence suggest that the 16S rRNA and tRNA(Ala) are transcribed as a single unit, tRNA(Ile) is produced as an individual transcript and the 23S and 5S rDNAs are co-transcribed. Several of the features of this rDNA organization are unique, not having been described previously in any other eubacteria.

Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans lesions.

Current laboratory diagnosis of Lyme disease relies on tests for the detection of antibodies to Borrelia burgdorferi, the etiologic agent of the disease. These tests are often unreliable because of a lack of sensitivity and specificity and test-to-test variability. The purpose of this study was to evaluate the sensitivity and specificity of polymerase chain reaction (PCR) amplification for detection of B. burgdorferi in skin biopsy specimens. Forty-six 2-mm skin biopsy samples were obtained from 44 patients with a clinical diagnosis of erythema migrans, 9 of whom were receiving antibiotic therapy at the time of biopsy. Specimens were ground in BSK medium with separate aliquots taken for culture and PCR. Of the specimens from the untreated group, 57% (21 of 37) were positive by culture and 22% (8 of 37) were culture negative; 22% (8 of 37) of the cultures were uninformative because of contamination. By comparison, 22 (59%) of 37 specimens were positive by PCR amplification. Of 21 culture-positive samples, 13 (62%) were also positive by PCR analysis. Thus, the sensitivity of the PCR was 59 to 62%, based on either a clinical or cultural diagnosis of untreated Lyme disease. None of the nine specimens from antibiotic-treated patients grew in culture, whereas two of the nine were positive by PCR analysis. Given the complexity and time required for culture, PCR is a promising technique for the diagnosis of early Lyme disease.

rRNA gene organization in the Lyme disease spirochete, Borrelia burgdorferi.

Lyme disease is the most common vector-borne disease in the United States. The causative agent is the spirochete Borrelia burgdorferi. The copy number and organization of the genes encoding the rRNAs of this organism were determined. There is a single gene for 16S rRNA and two copies each of the 23S rRNA and 5S rRNA genes. All of the genes are located within a chromosomal fragment of approximately 9.5 to 10.0 kb. The 23S and 5S rRNA genes are tandemly duplicated in the order 23S-5S-23S-5S and are apparently not linked to the 16S rRNA gene, which is situated over 2 kb upstream from the 23S-5S duplication. The individual copies of the 23S-5S duplication are separated by a 182-bp spacer. Within each 23S-5S unit, an identical 22-bp spacer separates the 23S and 5S rRNA sequences from each other. The genome organization of the 23S-5S gene cluster in a number of different B. burgdorferi isolates obtained at a number of different geographical locations, as well as in several other species of Borrelia, was investigated. All isolates of B. burgdorferi tested displayed the tandem duplication, whereas the closely related species B. hermsii, B. anserina, and B. turicatae all contained a single copy of each of the genes. In addition, different geographical isolates of B. burgdorferi can be differentiated on the basis of a restriction fragment length polymorphism associated with the 23S-5S gene cluster. This polymorphism can be a useful tool for the determination of genetic relatedness between different isolates of B. burgdorferi.

5S-RNA genes of barley are located on the second chromosome.

The genes coding for 5S RNA in barley were cloned, sequenced, and their cluster was assigned to chromosome 2 using wheat-barley chromosome addition lines. High-resolution gel-electrophoresis of DNA and subsequent hybridization revealed new details of the organization of 5S DNA both in wheat and barley. The in situ hybridization of the cloned 5S gene with triploid endosperm nuclei also suggests that these genes are located in a single locus.