Measurement of the sensitivity of different commercial assays in the diagnosis of CMV infection in pregnancy.

To evaluate the performance of different commercial assays for the detection of recent cytomegalovirus (CMV) in pregnancy, the sensitivity and specificity of assays for CMV-specific IgM antibodies were compared. Routine specimens from pregnant women were screened for CMV IgM using the Abbott AxSYM assay. Sera that were reactive according to AxSYM were further tested for IgM by other commercial assays.

Detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in Rome, Italy.

Detection of a broad number of respiratory viruses is not undertaken currently for the diagnosis of acute respiratory infection due to the large and always increasing list of pathogens involved. A 1-year study was undertaken on children hospitalized consecutively for acute respiratory infection in a Pediatric Department in Rome to characterize the viruses involved. Two hundred twenty-seven children were enrolled in the study with a diagnosis of asthma, bronchiolitis, bronchopneumonia, or laringo-tracheo bronchitis.

The effects of prolonged treatment with zidovudine, lamivudine, and abacavir on a T-lymphoblastoid cell line.

A human T-lymphoblastoid cell line that is resistant to the antiviral activity of zidovudine (ZDV) and moderately resistant to lamivudine (3TC) has been obtained as a result of prolonged treatment with a combination of three nucleoside analogues (NA), ZDV, 3TC, and abacavir (ABV). These cells, called CEM(ZLA), are fully sensitive to ABV. The cellular resistance of the CEM(ZLA) cells to ZDV correlates with significant reductions in thymidine kinase (TK) activity and in the amount of intracellular TK protein.

A novel assay to detect low-titred antibodies to interferon beta in multiple sclerosis patients.

Neutralizing antibodies (NAbs) may compromise interferon (IFN) clinical efficacy in patients with multiple sclerosis (MS) receiving IFN-beta treatment. When bioassays are used for anti-IFN-beta antibody detection, they are unable to discriminate between NAbs or other interfering substances with anti-IFN activity. Here we report the development of an anti-IFN-beta Western blot method that facilitates the detection of IFN low-titred antibodies and characterizes such low neutralizing activity as specifically due to the presence of particular IFN antibodies.

Molecular cloning, expression and purification of protein 2A of hepatitis A virus.

Expression of the protein 2A of Hepatitis A virus (HAV), spanning amino acids 764 through 981 of the viral polyprotein results in a strong inhibition of cap-dependent translation (Maltese et al., 2000). However, the molecular mechanism responsible has remained unclear, in part because the HAV 2A protein was not available in amounts large enough to allow biological or structural studies.

Inhibition of cap-dependent gene expression induced by protein 2A of hepatitis A virus.

The viral protein 2A of hepatitis A virus (HAV) lacks the conserved 18 aa sequence found in other picornavirus proteases; hence, it is unclear whether the induction of CPE by culture-adapted HAV strains is due to 2A-mediated activity. Moreover, the cleavage sites and actual borders of HAV 2A are not known. Accordingly, a nested series of cDNA sequences encoding the segment of the HAV polyprotein (aa 760-1087) were linked to the 5'-UTR of poliovirus type 2 (Lansing strain) and inserted downstream of the gene encoding human growth hormone (GH).

'Primer alignment-and-extension': a novel mechanism of viral RNA recombination responsible for the rescue of inactivated poliovirus cDNA clones.

In the course of experiments designed to assess the potential role of alternative open reading frames (ORF) present in the 5'-terminal untranslated region (5'-UTR) of poliovirus type 1 (Mahoney strain) genomic RNA, we came across a double mutation that completely abrogated the infectivity of full-length cDNA clones. The infectivity was rescued in trans by cotransfecting COS-1 cells with short RNA transcripts of the wild-type 5'-UTR of poliovirus type 2 Lansing, provided a free 3'-OH was available. Direct sequencing of the viral RNA revealed that the infectious viruses recovered were recombinants Lansing/Mahoney, with variable points of 'crossing-over'.

Increased infectivity of hepatitis A virus cDNA clones with engineered 5'-terminal extra-cistronic sequences.

The genomic RNA of Hepatitis A virus (HAV), a picornavirus of the hepatovirus group, is a single-stranded molecule, ca. 7.5 kb in length of positive polarity.

Identification of an alternative open reading frame ("hidden gene"?) stringently required for infectivity of poliovirus cDNA clones.

Translation of the uncapped poliovirus RNA starts at the AUG triplet spanning positions 741-743, and proceeds uninterrupted for almost the entire length of the genome. Such a cap-independent mechanism of internal initiation of translation determines that a long, extra-cistronic region extends between the 5'-end and the main open reading frame (ORF). We have identified 10 short ORFs initiated by the alternative translation initiation codons ACG, AUA, and GUG in the 5'-terminal extra-cistronic region (5'-ECR) of poliovirus RNA.

The distance between the 3'-pyrimidine-rich tract and the AUG codon modulates internal initiation of translation of hepatitis A virus RNA.

Protein synthesis directed by hepatitis A virus (HAV) RNA is mediated by a mechanism involving the recognition of internal sequences. Two in-frame AUG codons initiate the long open reading frame (positions 734-736 and 740-742). The extra-cistronic region extending between the uncapped 5'-end and the ORF contains two pyrimidine-rich tracts (PRTs): one 12 nucleotides in length in the close vicinity of the initiator AUG, and a longer one between bases 94 and 140.

Mutational analysis of the 3'-terminal extra-cistronic region of poliovirus RNA: secondary structure is not the only requirement for minus strand RNA replication.

A series of mutations were introduced in the 3'-terminal untranslated region (3'-UTR) of full-length infectious poliovirus cDNA clones, and following transfection of COS-1 cells the ability of these constructs to generate viable viral particles and/or to support viral RNA synthesis was assayed. Substitution of the 3'-UTR of poliovirus RNA with the equivalent sequences of HAV RNA abrogated viral RNA replication, whereas the introduction of extended 'foreign' sequences between the open reading frame and the 3'-UTR was well tolerated. Point mutation that either destabilized the stem-and-loop structure or altered the sequence of the loop in domain 'Y' (nomenclature as per Pilipenko et al.

Role of the pyrimidine-rich tract on the translation of 'chimeric' polio-hepatitis A mRNAs with engineered 5'-terminal untranslated regions.

The 5'-terminal untranslated region (5'-UTR) of picornavirus RNA contains a series of cis-acting elements required for the internal initiation of translation, including a pyrimidine-rich tract (PRT), which in entero- and rhinoviruses is located about 20 nts upstream from a silent AUG triplet in the vicinity of the translation initiation site. In hepatitis A virus (HAV) RNA, the PRT is only 12 nts upstream from the legitimate AUG initiation codon, and a second, longer PRT in a region far removed from the translation initiation site. This 5'-distal PRT includes a 'core' sequence 80% homologous to the PRT of poliovirus RNA.

5' UTR of hepatitis A virus RNA: mutations in the 5'-most pyrimidine-rich tract reduce its ability to direct internal initiation of translation.

The 5'-terminal untranslated region (5' UTR) of the uncapped hepatitis A virus (HAV) RNA contains two pyrimidine-rich sequences; one about 20 nucleotides (nt) in length in the vicinity of the AUG initiation codon (nt 706-726), and a longer one (about 40 nt) encompassing nt 100 to 140. The latter includes a 13 nt 'core' sequence (positions 126-138 in the HM175 strain) which is 80% identical to the pyrimidine-rich tract of poliovirus type 1 RNA (Mahoney strain). Representative cDNAs of the entire 5' UTR of HAV RNA were inserted in the intercistronic region of the bi-cistronic plasmid pSV-GH/CAT between the genes coding for the human growth hormone (GH) and bacterial chloramphenicol acetyltransferase (CAT).

Isolation and molecular cloning of a fast-growing strain of human hepatitis A virus from its double-stranded replicative form.

A fast-growing strain of human hepatitis A virus was selected and characterized. The virus has the unusual property of developing a strong cytopathic effect in tissue culture in 7 to 10 days. Sequences of the viral genome were cloned into recombinant plasmids with the double-stranded replicative form as a template for the reverse transcription of cDNA.

Genomic RNA of mengovirus. VI. Translation of its two cistrons in lysates of interferon-treated cells.

The addition of low levels (40 ng/ml) of the synthetic double-stranded polyribonucleotide poly I:C to lysates of interferon-treated L-cells resulted in a strong inhibition (70 to 75%) of the in vitro translation of mengovirus RNA. Under these conditions, the rates of incorporation of [35S]methionine or formyl-[35S]methionine were depressed to a comparable extent. The sequences of mengovirus RNA recognized by ribosomes of interferon-treated cells at initiation of translation were compared with those present in initiation complexes formed by ribosomes of untreated controls.