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PubMed1985

Expression of Sendai virus defective-interfering genomes with internal deletions.

Sendai virus strain 7 has been shown to contain four defective interfering (DI) RNA species in which both genome termini and various adjacent fragments of the 3'-terminal NP gene and 5'-terminal L gene are represented, but most or all internal genes and gene boundaries are deleted. Previous sequence analyses of these mutant RNAs suggested that all four possessed the transcription initiation signal of the NP gene and the transcription termination signal of the L gene. The supposition that these signals should specify transcripts has now been supported by oligo(dT) selection of four DI 7 specific RNA species that had apparent molecular weights slightly lower than each DI genome. DI RNA 7a, which contains the entire NP gene, except for two U residues at the end of the poly(A) initiation signal, appeared to be transcribed solely as a readthrough product. Since DI RNA 7a contains the entire NP protein-coding sequence and DI RNAs 7c and 7d contain fragments of it, whereas DI RNA 7b is devoid of it, only transcripts of RNAs 7c and 7d were expected to specify fusion proteins containing NP gene-specific sequences. A strain 7-induced protein that reacted with monoclonal antibodies against the NP protein had the 33,000 Mr size appropriate for the translation product predicted by the sequence of RNA 7d. Other proteins of lower molecular weight were seen only in cells infected by strain 7, but they did not react with NP-specific antibody and their translation in vitro was not blocked by hybridization to an NP gene-specific oligonucleotide. Therefore, at least some of these proteins may be cellular products induced by DI virus infection. These DI transcripts and translation products may influence interference with replication of the parental helper virus.

Antibodies, Monoclonal↗
PubMed2002

Viable human cytomegalovirus recombinant virus with an internal deletion of the IE2 86 gene affects late stages of viral replication.

Using bacterial artificial chromosome (BAC) technology, we have constructed and characterized a human cytomegalovirus recombinant virus with a mutation in the exon specific for the major immediate-early region 2 (IE2) gene product. The resulting IE2 86-kDa protein (IE2 86) has an internal deletion of amino acids 136 to 290 and is fused at the carboxy terminus to enhanced green fluorescent protein (EGFP). The deletion also removes the promoter and initiator methionine for the p40 form of IE2 and initiator methionine for the p60 form of the protein, and therefore, these late gene products are not produced. The mutant virus IE2 86 Delta SX-EGFP is viable but exhibits altered growth characteristics in tissue culture compared with a full-length wild-type (wt) IE2 86-EGFP virus or a revertant virus. When cells are infected with the mutant virus at a low multiplicity of infection (MOI), there is a marked delay in the production of infectious virus. This is associated with slower cell-to-cell spread of the virus. By immunofluorescence and Western blot analyses, we show that the early steps in the replication of the mutant virus are comparable to those for the wt. Although there is significantly less IE2 protein in the cells infected with the mutant, there is only a modest lag in the initial accumulation of IE1 72 and viral early proteins, and viral DNA replication proceeds normally. The mutation also has only a small effect on the synthesis of the viral major capsid protein. The most notable molecular defect in the mutant virus infection is that the steady-state levels of the pp65 (UL83) and pp28 (UL99) matrix proteins are greatly reduced. In the case of UL83, but not UL99, there is also a corresponding decrease in the amount of mRNA present in cells infected with the mutant virus.

Cells, Cultured↗
PubMedDate not supplied

[Hepatitis C virus infection in internal medicine and infectious diseases departments in France. Preliminary results of a national epidemiological survey].

OBJECTIVES: In order to know the number and the main clinical features of patients with hepatitis C virus (HCV) infection, we conducted a national epidemiological study of patients followed in Internal Medicine and in Infectious Diseases departments in France. METHODS: For each patient with HCV infection seen between 13/03 and 13/04/1995 (in- or out-patients), a chart was completed. RESULTS: The response rate was about 30% with 2002 charts being analyzed. There were 59% males and 39% females, with a peak of frequency between 25 and 40 years, including 1241/2002 (62%) patients. Most patients were of French origin (85.7%). We found that 1436/2002 (72%) patients came from provinces of southern France and Paris. In 10% of cases the diagnosis was made during the study period. A route of transmission was identified in 86.4%, mainly drug abuse 60.4% and blood product transfusion 19.3%. At the time of diagnosis, main clinical features were: no symptom 47%, liver biological abnormalities 52%, cirrhosis 7.4%, cryoglobulinemia 2.7%, auto-immune disease 3%, hepatocellular carcinoma 0.85%. We frequently found a viral co-infection: HIV 56.1%; HBV 22.2%. A confirmation RIBA test was done in 67.8%, measurement of viremia in 33.4%, genotyping in 6.7% and liver biopsy in 28.7% of cases. Finally, 407 (20.3%) patients received a specific anti-HCV treatment. CONCLUSION: These preliminary results underscore: 1) the large percentage of the population with HCV infection followed outside of hepatogastroenterology departments in France. 2) the high probability that the HCV epidemic is underestimated as we noted 10% of new diagnosis during the study period. 3) the requirement for a multidisciplinary approach for these HCV patients.

Adult↗
PubMed2000

Vaccination with DNA encoding internal proteins of influenza virus does not require CD8(+) cytotoxic T lymphocytes: either CD4(+) or CD8(+) T cells can promote survival and recovery after challenge.

DNA vaccination offers the advantages of viral gene expression within host cells without the risks of infectious virus. Like viral vaccines, DNA vaccines encoding internal influenza virus proteins can induce immunity to conserved epitopes and so may defend the host against a broad range of viral variants. CD8(+) cytotoxic T lymphocytes (CTL) have been described as essential effectors in protection by influenza nucleoprotein (NP), although a lesser role of CD4(+) cells has been reported. We immunized mice with plasmids encoding influenza virus NP and matrix (M). NP + M DNA allowed B6 mice to survive otherwise lethal challenge infection, but did not protect B6-beta(2)m(-/-) mice defective in CD8(+) CTL. However, this does not prove CTL are required, because beta(2)m(-/-) mice have multiple immune abnormalities. We used acute T cell depletion in vivo to identify effectors critical for defense against challenge infection. Since lung lymphocytes are relevant to virus clearance, surface phenotypes and cytolytic activity of lung lymphocytes were analyzed in depleted animals, along with lethal challenge studies. Depletion of either CD4(+) or CD8(+) T cells in NP + M DNA-immunized BALB/c mice during the challenge period did not significantly decrease survival, while simultaneous depletion of CD4(+) and CD8(+) cells or depletion of all CD90(+) cells completely abrogated survival. We conclude that T cell immunity induced by NP + M DNA vaccination is responsible for immune defense, but CD8(+) T cells are not essential in the active response to this vaccination. Either CD4(+) or CD8(+) T cells can promote survival and recovery in the absence of the other subset.

Amino Acid Sequence↗
PubMed1988

Simultaneous determination of the level of antibodies to influenza virus surface and internal proteins by enzyme-linked immunosorbent assay.

Enzyme-linked immunosorbent assay (ELISA) has been adopted for simultaneous determination of the levels of antibodies to different influenza virus proteins in human sera with known haemagglutination-inhibition (HI) titre. Whole virus of serotypes H1N1 and H3N2, haemagglutinin (HA), matrix (M) and nucleoprotein (NP) proteins have been used as antigens. For detection of antibodies bound to the antigen, peroxidase labelled Staphylococcus protein A conjugate has been used. Correlation of the ELISA and HI titres of anti-HA antibody has been demonstrated. The use of isolated HA as antigen increased the specificity of ELISA. The analysis of human reconvalescent sera has shown that increase in the titre of antibodies to internal proteins does not always coincide with the increase of antibody level to HA. Out of 8 sera with significant increase of the HI titre to the H3 subtype 5 specimens showed 4-fold increase of antibody titre to NP protein. The antibody titre to M protein was elevated in 2 sera only, while 1 serum showed no rise of antibody response to the tested viral proteins.

Adolescent↗
PubMed1987

Sequences of the Epstein-Barr Virus (EBV) large internal repeat form the center of a 16-kilobase-pair palindrome of EBV (P3HR-1) heterogeneous DNA.

We have previously characterized several genomic rearrangements of Epstein-Barr virus (EBV) DNA contained in one of the defective EBV genomes harbored by the P3HR-1 (HR-1) line (H. B. Jenson, M. S. Rabson, and G. Miller, J. Virol. 58:475-486, 1986). One recombinant clone of heterogeneous DNA (het DNA) from this defective genome is an EcoRI fragment of 16 kilobase pairs (kbp) which is a palindrome. DNA digestion fragments specific for the center of this palindrome were present in cells which contained het DNA but not in cells which lacked het DNA. Thus, the palindrome was not an artifact of DNA cloning. The organization of the center of this palindrome was studied by DNA sequencing. The comparable region of the parental HR-1 genome was also studied by DNA sequencing. The central 3,495 base pairs (bp) of the palindrome were composed of sequences derived exclusively from internal repeat 1 of EBV, represented by BamHI W fragment. At each end of the central 3,495 hp was a symmetrical recombination with sequences of BamHI-Z, located more than 50 kbp away on the standard EBV genome. The central 3,495 bp were composed of an unduplicated 341 bp flanked by two perfect palindromic repeats of 1,577 bp. The 341-bp unique region was a portion of a 387-bp region of standard HR-1 BamHI-W which was identical to the central 387 bp of the palindrome. This central 387-bp region contained numerous stretches of dyad symmetry capable of forming a large stem-and-loop structure. The palindromic rearrangement had created two novel open reading frames in het DNA derived from standard HR-1 BamHI-W sequences. These two het DNA open reading frames had different amino termini but identical carboxy termini derived from the large open reading frame in standard HR-1 BamHI-W (HR-1 BWRF1). The BamHI-W sequences found in het DNA did not include either the TATA box of standard HR-1 BamHI-W or the exons which are present in the potentially polycistronic latent mRNAs encoding EBV nuclear antigens. These marked alterations in genomic structure may relate to the unique biologic properties of virus stocks containing het DNA by creation of new polypeptides or by formation or deletion of regulatory or functional signals.

Base Sequence↗
PubMed2002

Capsid protein C of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence.

Deletions ranging in size from 4 to 21 amino acid residues were introduced into the capsid protein of the flavivirus tick-borne encephalitis (TBE) virus. These deletions incrementally affected a hydrophobic domain which is present at the center of all flavivirus capsid protein sequences and part of which may form an amphipathic alpha-helix. In the context of the full-length TBE genome, the deletions did not measurably affect protein expression and up to a deletion length of 16 amino acid residues, corresponding to almost 17% of mature protein C, viable virus was recovered. This virus was strongly attenuated but highly immunogenic in adult mice, revealing capsid protein C as a new and attractive target for the directed attenuation of flaviviruses. Apparently, the larger deletions interfered with the correct assembly of infectious virus particles, and this disturbance of virion assembly is likely to be the molecular basis of attenuation. However, all of the mutants carrying large deletions produced substantial amounts of subviral particles, which as judged from density gradient analyses were identical to recombinant subviral particles as obtained by the expression of the surface proteins prM and E alone. The structural and functional flexibility of protein C revealed in this study and its predicted largely alpha-helical conformation are reminiscent of capsid proteins of other enveloped viruses, such as alphaviruses (N-terminal domain of the capsid protein), retroviruses, and hepadnaviruses and suggest that all of these may belong to a common structural class, which is fundamentally distinct from the classical beta-barrel structures of many icosahedral viral capsids. The possibility of attenuating flaviviruses by disturbing virus assembly and favoring the production of noninfectious but highly immunogenic subviral particles opens up a promising new avenue for the development of live flavivirus vaccines.

Amino Acid Sequence↗
PubMed1997

The internal ribosomal entry site of the encephalomyocarditis virus enables reliable coexpression of two transgenes in human primary T lymphocytes.

It is essential for the improvement of adoptive cell therapies to generate efficiently large populations of human primary T cells that reliably express a suicide gene conferring drug sensitivity, such as herpes simplex virus thymidine kinase (HSVtk). We show here that an optimized dicistronic vector containing the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) is functional in human primary. T lymphocytes that bear on average one integrated vector copy per cell. We demonstrate reliable coexpression of the marker NTP, an inactive mutant of the human low-affinity nerve growth factor receptor and HSVtk. In the dicistronic vector NIT, NTP is expressed as a cap-dependent marker and HSVtk as a nonselectable IRES-dependent gene. Cell-surface expression of NTP is sufficient to allow for the efficient and rapid enrichment of the transduced cells to high purity. Of these purified lymphocytes, 97 +/- 4% and 92 +/- 6% are selectively eliminated when cultured in the presence of 1.0 or 0.1 microM ganciclovic respectively, establishing that the EMCV IRES ensures efficient and sufficient expression of two genes in human primary T cells.

Animals↗
PubMed1997

Valaciclovir versus acyclovir in the treatment of first-episode genital herpes infection. Results of an international, multicenter, double-blind, randomized clinical trial. The Valaciclovir International Herpes Simplex Virus Study Group.

BACKGROUND AND OBJECTIVES: Valaciclovir, the L-valine ester prodrug of acyclovir, is much better absorbed than acyclovir and produces acyclovir exposures three to five times those attainable with the parent drug. GOALS: To determine whether the improved bioavailability of valaciclovir and a more convenient, less frequent dose regimen can maintain the clinical efficacy previously demonstrated for acyclovir. STUDY DESIGN: This was an international, multicenter, randomized, double-blind clinical trial comparing 10-day regimens of valaciclovir (1000 mg, twice daily) and acyclovir (200 mg, 5 times daily) in the treatment of 643 otherwise healthy adults (> or = 18 years of age) with first-episode genital herpes. Patients were evaluated clinically and lesions were staged and cultured on days 1, 2, 3, 5, 7, 10, 14, and then twice weekly until healed. Blood for herpes serology tests was obtained on days 1 and 14; hematology and chemistry toxicity screening was done on days 1 and 7. RESULTS: Valaciclovir and acyclovir did not differ significantly in efficacy with respect to duration of viral shedding (hazard ratio, 1.00; 95% confidence interval [CI], 0.84-1.18), time to healing (hazard ratio, 1.08; 95% CI, 0.92-1.27), duration of pain (hazard ratio, 1.0; 95% CI, 0.85-1.18), and time to loss of all symptoms (hazard ratio, 1.02; 95% CI, 0.85-1.22). Patients with primary genital herpes (no preexisting antibody to either herpes simplex virus type at enrollment with seroconversion at day 14) had longer times to healing and longer duration of viral shedding and pain than patients with nonprimary first genital episodes. Adverse experiences were generally infrequent and mild and were comparable in the two treatment groups. CONCLUSIONS: Twice-daily valaciclovir proved as effective and well tolerated in the treatment of first-episode genital herpes as five-times-daily acyclovir. Valaciclovir provides a useful alternative to acyclovir with the advantage of a more convenient dosing regimen and the potential for improved compliance.

Acyclovir↗
PubMed2002

Diffraction quality crystals of PRD1, a 66-MDa dsDNA virus with an internal membrane.

It has proved difficult to obtain well diffracting single crystals of macromolecular complexes rich in lipid. We report here the path that has led to crystals of the bacteriophage PRD1, a particle containing approximately 2,000 protein subunits from 18 different protein species, around 10 of which are integral membrane proteins associated with a host-derived lipid bilayer of some 12,500 lipid molecules. These crystals are capable of diffracting X-rays to Bragg spacings below 4A. It is hoped that some lessons learned from PRD1 will be applicable to other lipidic systems and that these crystals will allow, as a proof of principle, the determination of the structure of the virus in terms of a detailed atomic model.

Bacteriophage PRD1↗