hCD46 receptor is not required for measles vaccine Schwarz strain replication in vivo: Type-I IFN is the species barrier in mice.

Measles virus has been successfully attenuated on chicken embryo cells to obtain a highly efficient and safe live attenuated vaccine, administered thus far to billions of children. Measles virus attenuation has long been described to involve a modification of cellular tropism with the use of human CD46 ubiquitous receptor. Nevertheless, the use of this receptor in vivo is not obvious. In this study we use four different mouse models to decipher the respective part of hCD46 receptor and type-I interferon response in measles host restriction. We observed that only type-I interferon restricts viral replication of attenuated MV Schwarz strain in mice, independently of the presence of hCD46 receptor. By comparing measles virus immunogenicity in the different models, we confirmed that there was no impact on the absence of this receptor on the immune response. Therefore, we propose to simplify the mouse model.

ViralORFeome: an integrated database to generate a versatile collection of viral ORFs.

Large collections of protein-encoding open reading frames (ORFs) established in a versatile recombination-based cloning system have been instrumental to study protein functions in high-throughput assays. Such 'ORFeome' resources have been developed for several organisms but in virology, plasmid collections covering a significant fraction of the virosphere are still needed. In this perspective, we present ViralORFeome 1.0 (http://www.viralorfeome.com), an open-access database and management system that provides an integrated set of bioinformatic tools to clone viral ORFs in the Gateway(R) system. ViralORFeome provides a convenient interface to navigate through virus genome sequences, to design ORF-specific cloning primers, to validate the sequence of generated constructs and to browse established collections of virus ORFs. Most importantly, ViralORFeome has been designed to manage all possible variants or mutants of a given ORF so that the cloning procedure can be applied to any emerging virus strain. A subset of plasmid constructs generated with ViralORFeome platform has been tested with success for heterologous protein expression in different expression systems at proteome scale. ViralORFeome should provide our community with a framework to establish a large collection of virus ORF clones, an instrumental resource to determine functions, activities and binding partners of viral proteins.

Persistent measles virus infection of mouse neural cells lacking known human entry receptors.

AIMS: Infection of the mouse central nervous system with wild type (WT) and vaccine strains of measles virus (MV) results in lack of clinical signs and limited antigen detection. It is considered that cell entry receptors for these viruses are not present on murine neural cells and infection is restricted at cell entry. METHODS: To examine this hypothesis, virus antigen and caspase 3 expression (for apoptosis) was compared in primary mixed, neural cell cultures infected in vitro or prepared from mice infected intracerebrally with WT, vaccine or rodent neuroadapted viruses. Viral RNA levels were examined in mouse brain by nested and real-time reverse transcriptase polymerase chain reaction. RESULTS: WT and vaccine strains were demonstrated for the first time to infect murine oligodendrocytes in addition to neurones despite a lack of the known MV cell receptors. Unexpectedly, the percentage of cells positive for viral antigen was higher for WT MV than neuroadapted virus in both in vitro and ex vivo cultures. In the latter the percentage of positive cells increased with time after mouse infection. Viral RNA (total and mRNA) was detected in brain for up to 20 days, while cultures were negative for caspase 3 in WT and vaccine virus infections. CONCLUSIONS: WT and vaccine MV strains can use an endogenous cell entry receptor(s) or alternative virus uptake mechanism in murine neural cells. However, viral replication occurs at a low level and is associated with limited apoptosis. WT MV mouse infection may provide a model for the initial stages of persistent MV human central nervous system infections.

Sequence analysis of the large (L) polymerase gene and trailer of the peste des petits ruminants virus vaccine strain Nigeria 75/1: expression and use of the L protein in reverse genetics.

The large (L) polymerase gene and the 5'-terminal UTR of the genome of peste des petits ruminants virus (PPRV), vaccine strain Nigeria 75/1, were cloned and sequenced. The L protein was also expressed in eukaryotic cells and its polymerase activity was quantitatively measured in a PPR reverse genetics assay using a reporter minigenome. Comparative sequence analysis of this functional L gene with corresponding genes of other morbilliviruses showed a degree of conservation exceeding 70%. The multiple sequence alignment and the phylogenetic study of L gene discriminated the morbilliviruses in 6 clusters, which are more closely related to Tupaia and Henipaviruses than to other paramyxoviruses. Important protein domains and functional motifs of the L polymerase of the PPRV Nigeria 75/1 vaccine were also identified by using different bioinformatics tools.

[Live attenuated measles vaccine as a potential multivalent pediatric vaccination vector]. / Utilisation potentielle du vaccin vivant atténué contre la rougeole comme vecteur de vaccination pédiatrique multivalent.

Live attenuated RNA viruses make highly efficient vaccines. Among them, measles virus (MV) vaccine has been given to a very large number of children and shown to be highly efficacious and safe. MV vaccine induces a life-long immunity after a single or two low-dose injections. It is easily produced on a large scale in most countries and can be distributed at low cost. Reversion to pathogenicity has never been observed with this vaccine. For all these characteristics, MV vaccine might be a very promising vector to immunize children against both measles and other infectious agents such as HIV or flaviviruses, in the developing world. In this article, we describe recent data demonstrating the capacity of recombinant Schwarz measles virus to express proteins from Human Immunodeficiency or West Nile viruses, and to induce specific immune responses able, in the case of West Nile virus, to protect from an experimental challenge.

Lymphoid organs as a major reservoir for human T-cell leukemia virus type 1 in experimentally infected squirrel monkeys (Saimiri sciureus): provirus expression, persistence, and humoral and cellular immune responses.

The aim of this study was to investigate the distribution of human T-cell leukemia virus type 1 (HTLV-1) in various organs of serially sacrificed squirrel monkeys (Saimiri sciureus) in order to localize the reservoir of the virus and to evaluate the relationship between viral expression and the humoral or cellular immune response during infection. Six squirrel monkeys infected with HTLV-1 were sacrificed 6, 12, and 35 days and 3, 6, and 26 months after inoculation, and 20 organs and tissues were collected from each animal. PCR and reverse transcription-PCR (RT-PCR) were performed with gag and tax primers. Proviral DNA was detected by PCR in peripheral blood mononuclear cells (PBMCs) of monkeys sacrificed 6 days after inoculation and in PBMCs, spleens, and lymph nodes of monkeys sacrificed 12 and 35 days and 3, 6, and 26 months after inoculation. Furthermore, tax/rex mRNA was detected by RT-PCR in the PBMCs of two monkeys 8 to 12 days after inoculation and in the spleens and lymph nodes of the monkey sacrificed on day 12. In this animal, scattered HTLV-1 tax/rex mRNA-positive lymphocytes were detected by in situ hybridization in frozen sections of the spleen, around the germinal centers and close to the arterial capillaries. Anti-HTLV-1 cell-mediated immunity was evaluated at various times after inoculation. Anti-p40(Tax) and anti-Env cytolytic T-cell responses were detected 2 months after infection and remained detectable thereafter. When Tax peptides were used, this response appeared to be directed against various Tax epitopes. Our results indicate that squirrel monkeys represent a promising animal model for studying the early events of HTLV-1 infection and for evaluating candidate vaccines against HTLV-1.

Human T cell leukemia virus type I expression in salivary glands of infected patients.

Human T cell leukemia virus type I (HTLV-I) sequences were sought in labial salivary glands of patients with HTLV-I-associated myelopathy or tropical spastic paraparesis and of seropositive neurologically healthy carriers. HTLV-I proviral DNA was found by polymerase chain reaction amplification in DNA extracted from lip biopsies of every patient. Viral RNA was found by in situ hybridization in the acini epithelium, as well as in lymphocytic infiltrates. This observation suggests that HTLV-I expression in labial salivary glands could participate in the inflammatory lesions observed in these patients. Some seronegative patients with Sjögren's syndrome or dryness syndrome were also positive for viral transactivator tax DNA (41% in Martinique and 16% in non-HTLV-I-endemic region). Despite histologic signs of lymphocytic infiltration, no viral expression was found in the labial salivary glands of these patients.

Molecular mechanism of tumorigenesis in mice transgenic for the human T cell leukemia virus Tax gene.

The infection by human T lymphotropic virus type I is associated with adult T cell leukemia and several inflammatory degenerative disorders, including tropical spastic paraparesis. To investigate the role of the Tax protein in the development of diseases linked to human T lymphotropic virus type I infection, we generated two lines of transgenic mice carrying the tax gene under the control of the viral promoter. The expression of the transgene was low in these mice and was restricted to the central nervous system and testis. Mice from both lines developed various types of tumors, including fibrosarcomas and adenocarcinomas. Tax was expressed at a high level in fibrosarcomas and in cell lines derived from these tumors. In tumor-derived cells, the expression of Tax led to an increased degradation of IkappaB alpha and IkappaB beta and caused stable nuclear translocation of nuclear factor-kappaB. This translocation was essential for cell proliferation, as shown by expressing a nondegradable form of IkappaBbeta in these cells. Therefore, Tax-induced cell transformation in mice correlates with the degradation of IkappaB alpha and IkappaB beta and with the constitutive activation of NF-kappaB.

A search for human T-cell leukemia virus type I in the lesions of patients with tropical spastic paraparesis and polymyositis.

We searched for the presence of human T-cell leukemia virus type I (HTLV-I) sequences in central nervous system and muscle lesions of 3 patients with tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) and 3 patients with HTLV-I-associated polymyositis. Proviral DNA coding for the Tax protein was found by polymerase chain reaction amplification in DNA extracted from lesions of every patient with TSP/HAM or HTLV-I-associated polymyositis. In contrast, viral RNA was found occasionally by in situ hybridization in muscle lesions of some patients with polymyositis, but was never found in central nervous system lesions of TSP/HAM patients.

Chimeric Theiler's virus with altered tropism for the central nervous system.

Theiler's virus is a neurotropic murine picornavirus which, depending on the strain, causes either an acute encephalitis or a persistent demyelinating disease. Following intracranial inoculation, the demyelinating strains infect sequentially the grey matter of the brain, the grey matter of the spinal cord, and finally the white matter of the spinal cord, where they persist and cause chronic demyelination. The neurovirulent strains cause a generally fatal encephalitis with lytic infection of neurons. The study of chimeric Theiler's viruses, obtained by recombining the genomes of demyelinating and neurovirulent strains, has shown that the viral capsid contains determinants for persistence and demyelination. In this article we describe the recombinant virus R5, in which the capsid protein VP1 and a small portion of protein 2A come from the neurovirulent GDVII strain and the rest of the genome comes from the persistent DA strain. The capsid of virus R5 also contains one mutation at amino acid 34 of VP3 (Asn-->His). Virus R5 does not persist in the central nervous system (CNS) of immunocompetent SJL/J or BALB/c mice. However, it replicates efficiently and persists in the CNS of BALB/c nu/nu mice, showing that its growth in the CNS is not impaired. In BALB/c nu/nu mice, whereas virus DA causes mortality with large amounts of viral antigens in the white matter of the spinal cord, virus R5 does not kill the animals, persists in the neurons of the grey matter of the brain, and never reaches the white matter of the spinal cord. This phenotype is due to the chimerism of the capsid and/or to the mutation in VP3. These results indicate that the capsid plays an important role in the characteristic migration of Theiler's virus within the CNS.

Determinants of persistence and demyelination of the DA strain of Theiler's virus are found only in the VP1 gene.

The DA strain of Theiler's virus persists in the central nervous systems of mice and causes chronic inflammation and demyelination. The GDVII strain, on the other hand, causes an acute encephalitis that kills the host in a matter of days. We constructed a series of recombinants between two infectious cDNA clones of the genomes of DA and GDVII viruses. Analysis of the phenotypes of the recombinant viruses yielded the following results. (i) Determinants of persistence and demyelination are found only in the VP1 capsid protein of DA virus. (ii) Whereas the VP1 capsid protein of DA virus is able to fully attenuate the neurovirulence of GDVII virus and to allow the chimeric virus to persist and demyelinate, the VP1 capsid protein of GDVII virus is unable to render DA virus neurovirulent. (iii) The mere attenuation of the neurovirulence of GDVII virus does not allow it to persist and demyelinate.

Genetic mapping of the ability of Theiler's virus to persist and demyelinate.

Theiler's virus, a murine picornavirus, is responsible for two different types of disease: strains DA, BeAn, and WW persist for more than a year in the white matter of the central nervous system and cause primary demyelination; strains GDVII and FA, on the other hand, cause an acute encephalitis that kills the host in a matter of days. To map the regions of the viral genome responsible for persistence and demyelination, cDNA clones of the entire genomes of the DA and GDVII strains were constructed and cloned into Bluescript plasmid (A. McAllister, F. Tangy, C. Aubert, and M. Brahic, Microb. Pathogen. 7:381-388, 1989; F. Tangy, A. McAllister, and M. Brahic, J. Virol. 63:1101-1106, 1989). We constructed chimeric viruses obtained by exchanging regions between the cDNA clones. Analysis of the disease phenotypes produced by the chimeric viruses allowed us to map persistence and demyelination to a genome segment coding for the VP1 capsid protein and 27 amino acids of protein 2A.

Molecular cloning of the complete genome of Theiler's virus, strain DA, and production of infectious transcripts.

We constructed a complete cDNA clone of the genome of Theiler's virus strain DA in a Bluescript plasmid. This recombinant plasmid, called pTMDA, was used to synthesize full length RNA transcripts of the viral insert. The RNA was infectious for BHK cells. Virus R1-DA, obtained from transfected BHK cells, caused the biphasic disease classically observed with this strain of Theiler's virus. SJL/J mice did not show clinical symptoms during the first week following intracranial inoculation, although viral antigens were found in a few neurons of brain and spinal cord. By 45 days post-inoculation, the mice had developed a chronic demyelinating disease and viral RNA and antigens could be found only in spinal cord white matter in areas surrounded by inflammatory infiltrates. At this stage no RNA or antigens were found in neurons. Therefore the phenotype of R1-DA was indistinguishable from that of genuine DA Theiler's virus.

Molecular cloning of the complete genome of strain GDVII of Theiler's virus and production of infectious transcripts.

We constructed a complete cDNA clone of strain GDVII of Theiler's virus in Bluescript plasmid. This recombinant plasmid, called pTMGDVII, was used to synthesize full-length RNA transcripts of the viral insert. This RNA was infectious for BHK cells. Virus R1-GDVII, obtained from transfected BHK cells, caused rapidly fatal encephalomyelitis in BALB/c mice. High amounts of viral antigens were present in neurons. No antigens were found in white matter. Therefore, the phenotype of R1-GDVII was indistinguishable from that of genuine GDVII Theiler's virus.

Theiler's virus genome is closely related to that of encephalomyocarditis virus, the prototype cardiovirus.

Theiler's virus causes a persistent demyelinating infection of the mouse central nervous system. Our study of the molecular mechanism of persistence led us to sequence 1925 nucleotides located at the 3' end of the viral genome. We observed extensive homologies between this region and the corresponding region of encephalomyocarditis virus, the prototype cardiovirus, and only some homologies with the 3' ends of foot-and-mouth disease virus, rhinovirus, and poliovirus genomes.

Effects of cations, polyamines and other aminoglycosides on gentamicin C2. Binding to ribosomes from sensitive and resistant Escherichia coli strains.

Gentamicin C2 interacts cooperatively with ribosomes from a sensitive Escherichia coli strain in a multiphasic way with several classes of sites. It is shown that this binding is highly-dependent on Mg++ and natural endogenous polyamine concentrations. The differences observed between ribosomes from sensitive and resistant strains may be explained by the absence of specific cooperative gentamicin interactions with resistant ribosomes. The effects of other aminoglycoside antibiotics are discussed in terms of structure-activity relationships.

Mechanism of action of gentamicin components. Characteristics of their binding to Escherichia coli ribosomes.

The binding of gentamicin (Gm) to Escherichia coli ribosomes and ribosomal subunits has been studied. By means of equilibrium dialysis and of statistical interpretation of the data it was found that [3H]gentamicin C2 and 6'-N-[3H]methylgentamicin C1a interact with three classes of sites on tight-coupled 70-S species: a first class concerning the tight and non-cooperative interaction with one drug molecule (Kd = 0.6 microM), a second class in which about five Gm molecules bind cooperatively (mean Kd = 10 microM), and a third class of very high capacity in which up to 70 drug molecules may interact. The extreme cooperativity of the third class of sites induces such an increase in the affinity for Gm that it may allow the shift of molecules already bound from high-affinity sites towards lower-affinity sites. The alteration of a ribosomal protein, L6, in a gentamicin-resistant mutant of E. coli abolished the multiclass and the cooperative aspects of ribosomes--gentamicin interaction. The large ribosomal subunits from E. coli MRE 600 strain interact cooperatively with Gm, whereas 50-S particles from the resistant mutant bind the drug in a diffuse way with high capacity and low affinity. The small subunits from both strains behave identically towards Gm. A good correlation is observed in comparing the gentamicin concentrations capable of saturating the different ribosomal classes of sites with concentrations inducing its multiphasic effects on protein synthesis.

Photo-induced labelling of Escherichia coli ribosomes by a tobramycin analog.

An [3H]azidobenzyl derivative of tobramycin, a 4,6-disubstituted 2-deoxystreptamine aminoglycoside, has been synthesized, and its ability to label Escherichia coli 70-S ribosomes under photoactivation has been studied. Two concentrations of the photolabel, corresponding to the saturation of the two classes of tobramycin sites on the ribosomes, were used. The results show that, at high antibiotic concentrations which induce maximal misreading during protein synthesis, most of the ribosomal proteins are labelled. At low antibiotic concentration, which results in the saturation of the first-class sites, a few proteins of both subunits are labelled, including L6, S4, S5, and, to a lesser extent, L2, L13 and S18. The 30-S subunit is, on the whole, labelled more efficiently than the 50-S subunit.

Have deoxystreptamine aminoglycoside antibiotics the same binding site on bacterial ribosomes?

(3H) Tobramycin was used as a probe to determine the relationship between the structure of aminoglycoside antibiotics and their ability to remove this drug from its higher affinity binding site on the ribosome. The dissacharide moieties (neamine, tobramine, gentamine) appeared to have a common binding site, whereas the kanosamine, garosamine and ribose moieties determined the specificity of this binding. Amikacin and butikacin behaved in an anomalous manner in spite of their close structural relationship to tobramycin.

Mechanism of action of aminoglycoside antibiotics. Binding studies of tobramycin and its 6'-N-acetyl derivative to the bacterial ribosome and its subunits.

6'-N-[14C]Acetyl-tobramycin and [3H]tobramycin were synthesized and their binding to Escherichia coli ribosomes and ribosomal subunits studied using equilibrium dialysis. THE 70-S ribosome, as well as its 50-S and 30-S subunits, bound tightly to 6'-N-acetyl-tobramycin. The binding of [3H]tobramycin to ribosomes was quite different. The 70-S ribosome was observed to possess several classes of binding sites; of these, one was determined to be of higher affinity and lower capacity, the 6'-N-[14C]acetyl-tobramycin site. The isotopic dilution method was used to define the specificity of the interaction. The selective binding of 6'-N-[14C]acetyl-tobramycin was highly reversible by tobramycin, kanamycins A, B, C and neomycin, but not by streptomycin or erythromycin. Gentamicin C1a was a poor inhibitor. This suggested that either the kanosamin or garosamin rings might be determinant in the binding of these molecules, as well as the 6'-amino group.