Samir Mounir

Molecular analysis of the ORFs 3 to 7 of porcine reproductive and respiratory syndrome virus, Québec reference strain.

SummaryThe cDNA sequence of the 3′-terminal genomic region of the Québec IAF-exp91 strain of porcine reproductive and respiratory syndrome virus (PRRSV) was determined and compared to those of other reference strains from Europe (Lelystad virus) and US (ATCC VR2385, MN-1b). The sequence (2834 nucleotides) which encompassed ORFs 3 to 7 revealed extensive genomic variations between the Québec strain and Lelystad virus (LV), resulting from high number of base substitutions, additions and deletions. The ORFs 5, 3, and 7 seemed to be relatively the most variable; the predicted encoding products of the Québec and LV strains displayed only 52%, 54%, and 59% amino acid identities, respectively. Nevertheless, in vitro translation experiments of the structural genes (ORFs 5, 6, and 7) and radio-immunoprecipitation assays with extracellular virions gave results similar to those previously reported for LV. In contrast, close genomic relationships were demonstrated between Québec and US strains. Taking together, these results indicate that, although structurally similar, North American PRRSV strains belong to a genotype distinct from that of the LV, thus supporting previous findings that allowed to divide PRRSV isolates into two antigenic subgroups (U.S. and European).

Kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus

SummaryThe kinetics of appearance of antibodies directed to the major structural proteins N, M and E of porcine reproductive and respiratory syndrome virus (PRRSV) was followed in pigs naturally- and experimentally-exposed to the virus. Specific IgM antibody titers were first detected by indirect immunofluorescence (IIF) at the end of the first week of PRRSV infection, peaked by day 14 to 21 post-inoculation (p.i.), then rapidly decreased to undetectable levels by day 35 to 42 p.i. On the other hand, specific IgG antibody titers peaked by day 21 to 28 p.i. and remained unchanged to the end of the 6- or 9-week observation period; in addition, a persistent viremia was observed. Virus neutralizing (VN) antibody titers >8 were not detected until 3 to 4 weeks p.i. Taken together, the results obtained by Western blotting analyses using purified virus andE. coli-expressed ORFs 5 to 7 gene products, suggested that antibodies directed against the envelope E protein appear by day 7 p.i., whereas antibodies directed against the nucleocapsid N and membrane M proteins can only be detected by the end of the second week p.i. No correlation could be demonstrated between VN and IIF antibody titers, viremia, and viral protein specificities of circulating antibodies at various times p.i.

Human coronavirus gene expression in the brains of multiple sclerosis patients

Abstract Total RNA extracted from both white and gray matter of brain tissue from multiple sclerosis (MS) patients and controls was analyzed using a reverse transcription-polymerase chain reaction for the presence of the nucleic acid of human coronavirus (HCV) 229E and OC43, the two strains characterized to date and associated with respiratory infections. HCV-229E viral RNA was detectable in the central nervous system tissue of 4 of 11 MS patients and in none of 6 neurological and 5 normal controls. No HCV-OC43 nucleic acid was detected in any of the specimens. These results suggest a neurotropism on the part of the 229E strain of human coronavirus and underline the importance of further studies on its tissue distribution. The fact that it was detected only in tissue from MS patients illustrates the need for continued studies on the possible role of coronaviruses in the etiology of MS.

Hepatitis C viral IRES inhibition by phenazine and phenazine-like molecules.

An in vitro assay based on the expression of Fluci reporter gene under the translational control of HCV IRES was used to evaluate and screen compound libraries. A structure-activity relationship study on a phenazine hit was conducted. Our data suggest that an intact phenazine or phenazine-like core with two distal polar substitutions is crucial for potency.

Sequence analysis of the membrane protein gene of human coronavirus OC43 and evidence for O-glycosylation

The gene encoding the membrane (M) protein of the OC43 strain of human coronavirus (HCV-OC43) was amplified by a reverse transcription-polymerase chain reaction of viral RNA with HCV-OC43- and bovine coronavirus (BCV)-specific primers. The nucleotide sequence of the cloned 1.5 kb fragment revealed an open reading frame (ORF) of 690 nucleotides which was identified as the M protein gene from its homology to BCV. This ORF encodes a protein of 230 amino acids with an M(r) of 26416. The gene is preceded by the motif UCCAAAC, analogous to the consensus coronavirus transcription initiation sequence. The M protein of HCV-OC43 shows features typical of all coronavirus M proteins studied: a hydrophilic, presumably external N terminus including about 10% of the protein, and a potential N-glycosylation site followed by three major hydrophobic transmembrane domains. The amino acid sequence of the M protein of HCV-OC43 has 94% identity with that of the Mebus strain of BCV, and also contains six potential O-glycosylation sites in the exposed N-terminal domain. Indeed, the glycosylation of the M protein was not inhibited in the presence of tunicamycin, which is indicative of O-glycosylation, as previously reported for BCV and murine hepatitis virus. Virions released from tunicamycin-treated cells contained the M glycoprotein but were devoid of both peplomer (S) and haemagglutinin-esterase (HE) proteins. Thus, inhibition of the N-glycosylation of the S and HE structural proteins prevented their incorporation into progeny virions, an indication that they are dispensable for virion morphogenesis, unlike the M protein.

Molecular characterization of the S protein gene of human coronavirus OC43

The gene encoding the spike protein of the OC43 strain of human coronavirus (HCV-OC43) was cloned and sequenced. The complete nucleotide sequence revealed an open reading frame of 4062 nucleotides encoding a protein of 1353 amino acids with a predicted M(r) of 150,078. Structural features include 22 N-glycosylation sites, an N-terminal hydrophobic signal sequence of 17 amino acids, an hydrophilic cysteine-rich sequence of 35 amino acids near the C terminus, and a potential proteolytic cleavage site (RRSR) between amino acid residues 758 and 759, yielding S1 and S2 segments of 84,730 and 65,366 M(r), respectively. The predicted amino acid sequence of the spike protein of HCV-OC43 has 91% identity with that of the Mebus strain of bovine coronavirus, revealing more sequence divergence in the putative bulbous part (S1) than in the predicted stem region (S2).

Phosphorothioate Di- and Trinucleotides as a Novel Class of Anti-Hepatitis B Virus Agents

ABSTRACT Several nucleoside analogs are under clinical development for use against hepatitis B virus (HBV). Lamivudine (3TC), a nucleoside analog, and adefovir dipivoxil (ADV), an acyclonucleotide analog, are clinically approved. However, long-term treatment can induce viral resistance, and following the cessation of therapy, viral rebound is frequently observed. There continues to be a need for new antiviral agents with novel mechanisms of action. A library of more than 600 di- and trinucleotide compounds synthesized by parallel synthesis using a combinatorial strategy was screened for potential inhibitors of HBV replication using the chronically HBV-producing cell line 2.2.15. Through an iterative process of synthesis, lead optimization, and screening, three analogs were identified as potent inhibitors of HBV replication: dinucleotides ORI-7246 (drug concentration at which a 10-fold reduction of HBV DNA was observed [EC90], 1.4 μM) and ORI-9020 (EC90, 1.2 μM) and trinucleotide ORI-7170 (EC90, 7.2 μM). These analogs inhibited the replication of both strands of HBV DNA. No suppression of HBV protein synthesis or intracellular core particle formation by these analogs was observed. No inhibition of HBV DNA strand elongation by the analogs or their 5′-triphosphate versions was apparent in in vitro polymerase assays. Although the exact mechanism of action is not yet identified, present data are consistent with an inhibition of the HBV reverse transcriptase-directed priming step prior to elongation of the first viral DNA strand. In transient-transfection assays, these analogs inhibited the replication of 3TC-resistant HBV. Synergistic interactions in combination treatments between the analogs and either 3TC or ADV were observed. These compounds represent a novel class of anti-HBV molecules and warrant further investigation as potential therapeutic agents.

Inhibition of tumor growth with doxorubicin in a new orthotopically implanted human hepatocellular carcinoma model.

A number of human xenograft orthotopic models of hepatocellular carcinoma (HCC) have been previously established by growing histologically-intact patient specimens in nude mice. However, the availability of HBV and HCV negative human hepatocellular carcinoma specimens is scarce and the pattern of tumor growth in nude mice varies depending on the tumor type. In the present study, we have established a reproducible xenograft orthotopic model using a human HCC cell line designated HuT7-3 that was derived from two rounds of subcloning of the parental Huh-7 cell line. The tumor growth rate of the HuT7-3 cell line, grown at a primary subcutaneous site, was markedly higher than that of the Huh-7 parental cell line or the human hepatoblastoma Hep-G2 cell line. Furthermore, we have shown that doxorubicin, when administered intravenously, is efficient in inhibiting the development of subcutaneous tumor but leads to the regression of the orthotopic human HCC. Consequently, this novel HCC xenograft orthotopic model can be used for the evaluation of antitumor drugs.

Parallel solid-phase synthesis of nucleoside phosphoramidate libraries.

Combinatorial chemistry is playing an increasingly prominent role in the process of drug discovery. A nucleic acid-based (NAB) scaffold can be engineered to create functional group and topological diversity in a library. Described herein is the parallel solid-phase synthesis of combinatorial libraries of nucleoside phosphoramidates, and the first evaluation of antiviral activity against hepatitis B virus (HBV).

Neurotropism of Human Coronavirus 229E

The 299E prototype strain of human coronavirus (HCV-229E) has so far been mainly associated with infections of the respiratory tract. In the present study, we show evidence for infection of the central nervous system (CNS) by HCV-229E, both in vitro and in vivo. Various human cell lines of CNS origin were tested for their susceptibility to infection by HCV-229E. Production of viral antigens was monitored by indirect immunofluorescence with monoclonal antibodies and infectious progeny virions by plaque assay on the L132 human embryonic lung cell line. The SK-N-SH neuroblastoma and H4 neuroglioma cell lines were highly susceptible to infection. The U-87 MG and U-373 MG astrocytoma cell lines were also infectable by HCV-229E. We could also demonstrate infection of the MO3.13 cell line, which was established by fusion of human oligodendrocytes with a thioguanine-resistant mutant of the TE671 (RD) human rhabdomyosarcoma cell line. An apparently more extensive infection of the MO3.13 cells, when compared to the parental cells, supports the notion that human oligodendrocytes are differentially susceptible to infection by this virus. We also tested for HCV-229E gene expression in pathological brain specimens. For that purpose, we developed a reverse transcription-polymerase chain reaction (RT-PCR) assay to amplify a portion of the mRNA encoding the viral nucleocapsid protein. Using stringent laboratory conditions, viral RNA was detectable in brain tissue of 4 of 11 multiple sclerosis patients and none of 6 neurological and 5 normal controls.(ABSTRACT TRUNCATED AT 250 WORDS)