ABSTRACT
In the present study, the effect of combining anti-influenza drugs active at different steps of the influenza virus replication cycle, oseltamivir as a neuraminidase (NA) inhibitor and amantadine targeting M2 protein, was investigated in vivo by oral administration in a mouse model of aerosol influenza virus infection and in vitro in MDCK cells. In mice, doses of oseltamivir and amantadine providing 50-60% survival against A/Hongkong/1/68 (H3N2) or A/PR/8/34 (H1N1) were capable of conferring complete protection when used simultaneously, suggesting that increased inhibition of influenza virus replication by combining oseltamivir and amantadine in vitro translates into protection from lethal infection of mice. The combination of amantadine with oseltamivir required 15-fold less oseltamivir than monotherapy to confer complete protection against lethal aerosol influenza virus infection. Remarkably, amantadine-based combination chemoprophylaxis was even effective against amantadine-resistant A/PR/8/34 influenza virus. Thus, combination chemotherapy may be more efficacious than monotherapy against newly emerging Influenza A subtypes.
Subject(s)
Amantadine/therapeutic use , Antiviral Agents/therapeutic use , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H3N2 Subtype/drug effects , Orthomyxoviridae Infections/prevention & control , Oseltamivir/therapeutic use , Amantadine/administration & dosage , Animals , Antiviral Agents/administration & dosage , Cell Line , Dogs , Dose-Response Relationship, Drug , Drug Combinations , Female , Mice , Mice, Inbred BALB C , Oseltamivir/administration & dosageABSTRACT
Porcine circovirus type 1 (PCV1) encodes two major ORFs. The cap gene comprises the major structural protein of PCV, the rep gene specifies Rep and Rep', which are both essential for initiating the replication of the viral DNA. Rep corresponds to the full-length protein, whereas Rep' is a truncated splice product that is frame-shifted in its C-terminal sequence. In this study, the cellular localization of PCV1-encoded proteins was investigated by immune fluorescence techniques using antibodies against Rep, Rep' and Cap and by expression of viral proteins fused to green and red fluorescence proteins. Rep and Rep' protein co-localized in the nucleus of infected cells as well as in cells transfected with plasmids expressing Rep and Rep' fused to fluorescence proteins, but no signal was seen in the nucleoli. Rep and Rep' carry three potential nuclear localization signals in their identical N-termini, and the contribution of these motifs to nuclear import was experimentally dissected. In contrast to the rep gene products, the localization of the Cap protein varied. While the Cap protein was restricted to the nucleoli in plasmid-transfected cells and was also localized in the nucleoli at an early stage of PCV1 infection, it was seen in the nucleoplasm and the cytoplasm later in infection, suggesting that a shuttling between distinct cellular compartments occurs.
Subject(s)
Cell Nucleus/chemistry , Circovirus/physiology , Viral Proteins/analysis , Amino Acid Sequence , Animals , Cell Line , Cell Nucleolus/chemistry , Cytoplasm/chemistry , Fluorescent Antibody Technique , Genes, Reporter , Green Fluorescent Proteins/analysis , Luminescent Proteins/analysis , Microscopy, Confocal , Molecular Sequence Data , Nuclear Localization Signals/physiology , Peptide Mapping , Recombinant Fusion Proteins/analysis , Swine , Red Fluorescent ProteinABSTRACT
Genome replication of Porcine circovirus type 1 (PCV1) relies upon expression of the full-length protein Rep and a spliced isoform (Rep'), and the presence of a 111-bp genomic fragment comprising the origin of replication. Using an electrophoretic mobility shift assay (EMSA), the capability of both Rep proteins to bind to partial fragments of the origin of replication of PCV1 was investigated in vitro. Both proteins formed complexes with double-stranded DNA origin fragments containing a stem-loop structure with a conserved nonamer and four hexamer repeats (5'-CGGCAG; H1 to H4). Use of truncated EMSA substrates identified minimal binding sites (MBS) for Rep and Rep' protein: The Rep binding site was mapped to the right leg of the stem-loop and the two inner hexamer repeats H1/H2, while binding of Rep' required only the presence of two hexamer repeats. Two differentially retarded complexes were observed with Rep protein, which presumably result from alternative binding to the MBS or to H3/4.
Subject(s)
Circovirus/genetics , DNA Helicases/metabolism , DNA, Viral/metabolism , DNA-Binding Proteins/metabolism , Replication Origin , Trans-Activators/metabolism , Animals , Base Sequence , Binding Sites , Circovirus/metabolism , DNA Helicases/biosynthesis , DNA Helicases/genetics , DNA, Single-Stranded , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Molecular Sequence Data , Mutagenesis , Nucleic Acid Conformation , Protein Binding , Protein Biosynthesis , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Protein Isoforms/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Swine/virology , Trans-Activators/biosynthesis , Trans-Activators/genetics , Transcription, GeneticABSTRACT
The complete DNA sequence of the 10-45 kbp HindIII B fragment of bovine herpesvirus type 4 (BoHV-4) was determined. This fragment contains nine complete and two incomplete open reading frames (ORFs), all of which are homologous to herpesvirus saimiri (HVS), Kaposi's Sarcoma-associated herpesvirus (HHV-8) and Epstein-Barr virus (EBV). Particularly, the arrangement of the gene for the terminase-related protein with the two coding exons 29a/29b is conserved among all herpesviruses sequenced to date. The intron carries the ORFs 30 to 33 in the opposite direction. Analysis by reverse transcription and polymerase chain reaction (PCR) of the transcript across the proposed splice junction of the ORF 29a/29b and subsequent sequence determination of the amplified product revealed the precise structure of the splice junction. Furthermore, the phylogenetic analysis of the 29a/29b protein and its counterparts in other herpesviruses revealed that BoHV-4 clustered in the genus Rhadinovirus of the subfamily Gammaherpesvirinae.
