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PubMed1984

Nucleotide sequence of the dsRNA genomic segment 7 of Simian 11 rotavirus.

A full length cDNA copy of dsRNA segment seven of Simian 11 rotavirus has been obtained by standard molecular cloning techniques. Segment seven codes for the non-structural viral protein NCVP4 and is 1104 nucleotides in length with putative 5'- and 3'- terminal non-coding regions of 25 and 134 residues respectively. The longest open reading frame of 315 codons extends from nucleotide 26 to 970 inclusive. However, the presence nearby of two other AUG codons makes it unclear which codon is used for initiation. The second AUG conforms to the Kozak consensus sequence and if utilised, would yield a protein 312 amino acids in length with a nett charge at pH7 of -2.5. Determination of the gene 7 sequence indicates that terminal sequence conservation among rotavirus gene segments is limited to three and two nucleotides at the 5' and 3' ends of the plus strand, respectively.

Amino Acid Sequence↗

PubMed1995

Direct measurement via phage titre of the dissociation constants in solution of fusion phage-substrate complexes.

Studies of interactions between filamentous fusion phage particles and protein or nucleic acid molecules have gained increasing importance with recent successes of screening techniques based upon random phage display libraries (biopanning). Since a number of different phage are usually obtained by biopanning, it is useful to compare quantitatively the binding affinities of individual phage for the substrate used for selection. A procedure is described for determination of relative dissociation constants (KdRel) between filamentous phage carrying peptide fusions to the coat protein gpIII and substrates in solution. This novel method is based on the measurement of phage titres. Phage selected from a random fusion phage library for binding to a monoclonal antibody or a viral structural protein exhibited KdRel values in the nanomolar and micromolar ranges for their respective substrates, thus validating the method over a wide range of binding affinities.

Bacteriophages↗

PubMed1999

Viral infections of the gastrointestinal tract.

Viral gastroenteritis is a major worldwide public health problem. The number of identified pathogens continues to increase, and characterization of the viral structures and functions must be inexorably pursued. Investigators have made considerable progress toward understanding the mechanisms of virus-cell interactions, host responses, and pathogenesis, but the application of this knowledge to improve disease treatment and prevention awaits further key discoveries. Despite deficiencies in knowledge of many aspects of these important issues, a vaccine for the most important pathogen, rotavirus, has been submitted to the US Food and Drug Administration for approval.

Journal Article↗

PubMed1993

Herpetic vaginitis in 1993.

Substantial advances have been made in our understanding of HSV and genital herpes. The molecular biology of viral structure and function and the pathogenesis of infection have been elucidated to a large degree, and epidemiologic investigations, based on reliable type-specific assays, have been done. Diagnostic techniques have been refined, and the value of therapy has been defined. Currently, management strategies for pregnant women and their neonates are based on data rather than hypothesis, and rational counseling is feasible. The next major advance in the field would be the availability of a safe and effective vaccine, the development and investigation of which is being pursued vigorously.

Acyclovir↗

PubMed1984

Monoclonal antibodies to the major capsid protein of human papillomavirus type 1.

Two stable monoclonal hybridoma cell lines secreting type-specific antibodies against the human papillomavirus type 1 (HPV-1) were isolated. The monoclonal antibodies detected HPV-1 antigens in frozen sections of HPV-1-induced warts, using immunofluorescence or immunoperoxidase techniques, and they reacted with HPV-1 particles in an immunodiffusion test. The two monoclonal antibodies recognized the major structural viral polypeptide, with a molecular weight of 54 000, and a minor polypeptide, with molecular weight of 76 000, in both the dissociated viral particles and in the wart extracts.

Antibodies, Monoclonal↗

PubMed1991

Pathogenesis of avian encephalomyelitis viruses.

The pathogenesis of a field strain, a vaccine strain and the egg-adapted Van Roekel strain of avian encephalomyelitis virus in susceptible chicken embryos and day-old chickens was investigated using enzyme-linked immunosorbent assays for the detection of virus-specific antibody and antigen. The Van Roekel strain was shown to be highly neurotropic whereas the field and vaccine strains were enterotropic. Radioimmuno-precipitation studies using Na125I-labelled purified virus failed to detect any differences in the composition of the structural viral proteins of each strain that could account for these differences. As expected, the field and vaccine strains were more efficient than the Van Roekel strain at inducing antibody following oral administration. Primary cultures of chicken embryo brain cells supported the growth of the Van Roekel strain to a much greater extent than the field and vaccine strains.

Animals↗

PubMed1992

Functional mapping of the rev-responsive element of human immunodeficiency virus type 2 (HIV-2): influence of HIV-2 envelope-encoding sequences on HIV-1 gp120 expression in the presence or absence of Rev.

The human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev stimulates expression of structural viral proteins via a target response element (RRE) located within gag-pol and env mRNAs. To analyse the HIV-2 Rev trans-activation effect on the expression of the envelope protein, we cloned a functionally active HIV-2 rev cDNA and showed that it contained four exons. Using transient expression assays, we mapped a 353 bp RRE fragment within the env gene of HIV-2 on which both HIV-1 and HIV-2 Rev could act. Interestingly, smaller fragments suppressed the use of additional splice sites within the env gene and caused envelope protein expression independent of Rev.

Base Sequence↗

PubMed1998

Immunological detection and mutational analysis of the RNA2-encoded nematode transmission proteins of pea early browning virus.

Pea early browning virus (PEBV) is transmitted between plants by root-feeding trichodorid nematodes. Mutagenesis studies have implicated two non-structural viral proteins in the transmission process. These two proteins [the 29 kDa ('29K') protein and the 23K protein] were expressed in bacteria and used to raise antibodies. In Western blotting experiments, the antibodies detected both of these virus proteins in leaves and roots of infected Nicotiana bethamiana and N. clevelandii plants. Periodate treatment of proteins transferred to nitrocellulose membranes suggested that the PEBV 23K protein may be glycosylated. A PEBV mutant was constructed lacking the complete 23K coding sequence. The mutant was able systemically to infect Nicotiana spp. but caused striking chlorotic ringspot leaf symptoms and stunting of both leaves and roots. These symptoms were absent in plants doubly-infected with the mutant and wild-type PEBV. The 23K gene deletion mutant was transmitted by nematodes at a much reduced frequency compared to wild-type virus, indicating that the 23K protein is involved in but not essential for vector transmission. Western immuno-blot and ELISA experiments revealed that the reduction in the nematode-transmissibility of PEBV carrying mutations in the 23K gene did not result from interference in the expression of the 29K transmission protein or from gross changes in the titre of virus in the roots of infected plants.

Animals↗

PubMed1998

Complementation of a fibre mutant adenovirus by packaging cell lines stably expressing the adenovirus type 5 fibre protein.

Adenovirus-based gene therapy vectors now in use cannot be targeted to specific cell types in vivo and are immunogenic, properties which limit their clinical utility. Improved vectors lacking the genes for viral structural proteins may overcome these limitations. We have developed cell lines which stably express the adenovirus type 5 (Ad5) fibre protein in its native trimeric form. These cells can complement an Ad5 mutant with a defect in the fibre gene, and are capable of incorporating the Ad5 fibre into particles of a different Ad serotype. As the fibre protein is responsible for the initial binding of virus to cells, packaging cell lines expressing different or modified fibre proteins will be useful in studying the mechanism by which adenovirus infects different cell types.

Adenoviridae↗

PubMed1997

Nucleic acid-based antiviral and gene therapy of chronic hepatitis B infection.

Persistent hepatitis B virus (HBV) infection often leads to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. There is a need to develop new antiviral approaches for the treatment of this disease. We have explored various nucleic acid-based strategies designed to inhibit HBV replication including: the use of antisense RNA and DNA constructs, DNA-based immunization techniques to stimulate broad-based cellular immune responses with particular emphasis on the generation of cytotoxic lymphocyte (CTL) activity to viral structural proteins, hammerhead ribozymes to cleave HBV pregenomic RNA in vitro and dominant negative HBV core mutant proteins as inhibitors of nucleocapsid formation within cells. In order to optimize these antiviral effects, various novel expression vectors have been developed to deliver such DNA constructs to cells. For example, adenoviral vectors carrying genes that encode for dominant negative proteins have been employed to transfect hepatocytes in vitro and in vivo. In addition, plasmid vectors have been produced to promote expression of HBV structural genes following injection into muscle cells as a means to stimulate the host's cellular and humoral immune response in the context of histocompatibility antigen (HLA) class I and II antigen presentation. These experimental approaches may have important implications for the generation of efficient antiviral effects during chronic HBV infection.

Antiviral Agents↗

PubMed1989

Comparison of human papillomavirus type 1 serotyping by monoclonal antibodies with genotyping by in situ hybridization of plantar warts.

Thirty plantar warts were analyzed for the presence of HPV-1 type-specific and PV genus-specific capsid antigens by immunofluorescence (IF) using monoclonal and polyclonal antibodies and type-specific HPV-1 DNA employing in situ hybridization methods. Fifteen of 30 plantar warts were positive by IF for PV genus-specific structural viral antigens. Thirteen of the 15 productively infected plantar warts expressed intranuclear HPV-1 type-specific capsid antigens and viral DNA, which were detected in the same distribution in each individual wart. The 2 productively infected plantar warts that did not react with HPV-1 type-specific MoAbs did not react with HPV-1 type-specific DNA by in situ hybridization. Thus, serotyping of HPV-1 capsid antigens by monoclonal antibodies is concordant with genotyping of HPV-1 viral DNA by in situ hybridization in productively infected plantar warts.

Antibodies, Monoclonal↗

PubMed1989

Relative repair of adenovirus damaged by sunlamp, UV and gamma-irradiation in Cockayne syndrome fibroblasts is different from that in xeroderma pigmentosum fibroblasts.

The DNA repair capacities of three unrelated Cockayne syndrome (CS) fibroblast strains were compared to that of three unrelated xeroderma pigmentosum (XP) strains for three different DNA damaging agents using a sensitive host cell reactivation (HCR) technique. Adenovirus type 2 (Ad 2) was treated with either UV light, gamma-rays or sunlamp-irradiation and subsequently assayed for its ability to form viral structural antigens (Vag) in the CS and XP strains using immunofluorescent straining. D37 values for the survival of Ad 2 Vag synthesis in the CS and XP strains, expressed as a percentage of those obtained in normal strains, were used as a measure of DNA repair capacity. Percent HCR values in the XP strains XP25RO, XP2BE and XP5BE respectively were lowest for UV (6, 14 and 6%), intermediate for sunlamp-irradiation (18, 32 and 10%) and highest for gamma-irradiation (65, 61 and 60%), whereas for the CS strains CS1BE, CS3BE and CS278CTO respectively, percent HCR values were lowest for UV (26, 30 and 34%), intermediate for gamma-irradiation (61, 64 and 69%) and near normal for sunlamp-irradiation (82, 73 and 89%). These results suggest that the 'spectrum of lesions' which is defectively repaired in CS is not the same as that which is defectively repaired in XP.

Adenoviruses, Human↗

PubMed1975

Structure of coat proteins in Pf1 and fd virions by laser raman spectroscopy.

Rama spectra of filamentous bacterial viruses are dominated by scattering from vibrations of the protein capsomers. The amino acid compositions of coat proteins in Pfl and fd strains are recognized by their different side-chain vibrational frequencies. However, the conformationally sensitive amide frequencies indicate that Pfl and fd coat proteins have the same alpha-helicial secondary structures. Viral DNA backbones do not exhibit the A-type geometry.

Animals↗

PubMed1987

Characterization and clinical association of antibody inhibitory to HIV reverse transcriptase activity.

Reverse transcriptase activity of the human immunodeficiency virus (HIV) was blocked in vitro by immunoglobulin G (IgG) derived from certain individuals infected with this retrovirus. A heterogeneous immune response for inhibition of enzyme function was noted. Catalytic activity was depressed by 50% or more with the use of 10 micrograms of IgG from 11 of 16 HIV-seropositive asymptomatic carriers, but from 0 of 8 seronegative controls and 2 of 12 patients with acquired immune deficiency syndrome (AIDS) or the AIDS-related complex (ARC). The inhibitor was confined to the F(ab')2 fragment. It was not directed against the poly(rA) X oligo(dT) template, nor against major envelope or structural viral antigens, and did not cross-react with bacterial, avian, or other mammalian DNA polymerases. It did not correlate with recognition of polymerase antigens by radioimmunoprecipitation. Loss of this inhibitor may be associated with development of clinical disease. Ten asymptomatic HIV-seropositive carriers with high titers of IgG antibodies to reverse transcriptase were followed for a mean of 3 years. All of four lost inhibitory capability prior to development of AIDS or ARC, while titers persist in the six who remain clinically healthy.

AIDS-Related Complex↗

PubMed1988

Virus-specific splicing inhibitor in extracts from cells infected with HIV-1.

Human immunodeficiency virus type 1 (HIV-1), in contrast with most other retroviruses, encodes trans-regulatory proteins for virus gene expression. It is shown in this study, by means of an in vitro splicing system, that nuclear extracts obtained from cells infected with HIV-1 contain a factor (or factors) that specifically inhibits splicing of a synthetic SP6/HIV pre-messenger RNA (pre-mRNA)-containing donor and acceptor splice sites in the coding region for the envelope protein. It is also shown that the SP6/HIV pre-mRNA is not capable of assembly in a ribonucleoprotein complex, spliceosome, in extracts from infected cells. These findings raise the possibility that specific inhibition of pre-mRNA splicing in the envelope protein coding region by HIV-1 trans-regulatory factors might be one control mechanism for efficient production of structural viral proteins and virion assembly.

Cell Nucleus↗

PubMed2000

Transmission of human T-cell lymphotropic virus type 1 tax to rabbits by tax-only-positive human cells.

The human T-cell lymphrotropic virus type 1 (HTLV-1) is causally related to adult T-cell leukemia and lymphoma and the neurodegenerative diseases tropical spastic paraparesis and HTLV-1-associated myelopathy. In the United States the prevalence of infection has been estimated to range from 0.016 to 0.1% on the basis of serologic tests for antibodies to the viral structural proteins. Blood from donors positive for antibodies to HTLV-1 or HTLV-2 is not used for transfusion. However, patients with the cutaneous T-cell lymphoma mycosis fungoides (MF) are HTLV-1 and -2 seronegative yet harbor proviral sequences identical to those that encode the HTLV-1 transactivating and transforming gene product p40tax in their peripheral blood mononuclear cells (PBMCs), and they usually have antibodies to p40(tax). Moreover, a study of 250 randomly selected blood donors revealed that approximately 8% of these seronegative individuals also had HTLV-1 tax sequences and antibodies to p40(tax), while they lacked sequences and antibodies related to gag, pol, or env. Thus, it seemed important to determine whether the "tax-only" state can be transmitted by transfusion. To this end, PBMCs from HTLV-1 and -2 seronegative tax-only-positive MF patients or from healthy tax-only-positive blood donors were injected into adult rabbits, an established animal model for HTLV-1 infection. The PBMCs of all injected rabbits became tax sequence positive. These observations suggest that HTLV-1 tax can be transmitted by tax-only-positive mononuclear cells.

Animals↗

PubMed2001

Candidate vaccine against botulinum neurotoxin serotype A derived from a Venezuelan equine encephalitis virus vector system.

A candidate vaccine against botulinum neurotoxin serotype A (BoNT/A) was developed by using a Venezuelan equine encephalitis (VEE) virus replicon vector. This vaccine vector is composed of a self-replicating RNA containing all of the VEE nonstructural genes and cis-acting elements and also a heterologous immunogen gene placed downstream of the subgenomic 26S promoter in place of the viral structural genes. In this study, the nontoxic 50-kDa carboxy-terminal fragment (H(C)) of the BoNT/A heavy chain was cloned into the replicon vector (H(C)-replicon). Cotransfection of BHK cells in vitro with the H(C)-replicon and two helper RNA molecules, the latter encoding all of the VEE structural proteins, resulted in the assembly and release of propagation-deficient, H(C) VEE replicon particles (H(C)-VRP). Cells infected with H(C)-VRP efficiently expressed this protein when analyzed by either immunofluorescence or by Western blot. To evaluate the immunogenicity of H(C)-VRP, mice were vaccinated with various doses of H(C)-VRP at different intervals. Mice inoculated subcutaneously with H(C)-VRP were protected from an intraperitoneal challenge of up to 100,000 50% lethal dose units of BoNT/A. Protection correlated directly with serum enzyme-linked immunosorbent assay titers to BoNT/A. The duration of the immunity achieved was tested at 6 months and at 1 year postvaccination, and mice challenged at these times remained refractory to challenge with BoNT/A.

Animals↗

PubMed1979

Restriction Maps of Five Autographa californica MNPV Variants, Trichoplusia ni MNPV, and Galleria mellonella MNPV DNAs with Endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI.

The restriction sites of Autographa californica nuclear polyhedrosis virus (AcMNPV) E2 DNA were mapped for the endonucleases SmaI, KpnI, BamHI, SacI, XhoI, and EcoRI. The restriction maps of four other AcMNPV variants, Trichoplusia ni (TnMNPV), and Galleria mellonella (GmMNPV) genomes were determined and compared to the endonuclease cleavage maps of AcMNPV E2 DNA. The viral structural polypeptides of AcMNPV variants S3, E2, S1, M3, and R9 were the same when analyzed by polyacrylamide gel electrophoresis. The major structural polypeptides of GmMNPV and TnMNPV had the same pattern in polyacrylamide gels as did AcMNPV structural polypeptides. GmMNPV and TnMNPV had several minor structural protein differences as compared with AcMNPV. AcMNPV variants, TnMNPV, and GmMNPV were distinct but with very similar genomes and protein structures.

Journal Article↗