Molecular diagnostic and genetic characterization of highly pathogenic viruses: application during Crimean-Congo haemorrhagic fever virus outbreaks in Eastern Europe and the Middle East.

Several haemorrhagic fevers are caused by highly pathogenic viruses that must be handled in Biosafety level 4 (BSL-4) containment. These zoonotic infections have an important impact on public health and the development of a rapid and differential diagnosis in case of outbreak in risk areas represents a critical priority. We have demonstrated the potential of a DNA resequencing microarray (PathogenID v2.0) for this purpose. The microarray was first validated in vitro using supernatants of cells infected with prototype strains from five different families of BSL-4 viruses (e.g. families Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae and Paramyxoviridae). RNA was amplified based on isothermal amplification by Phi29 polymerase before hybridization. We were able to detect and characterize Nipah virus and Crimean-Congo haemorrhagic fever virus (CCHFV) in the brains of experimentally infected animals. CCHFV was finally used as a paradigm for epidemics because of recent outbreaks in Turkey, Kosovo and Iran. Viral variants present in human sera were characterized by BLASTN analysis. Sensitivity was estimated to be 10(5) -10(6) PFU/mL of hybridized cDNA. Detection specificity was limited to viral sequences having ~13-14% of global divergence with the tiled sequence, or stretches of ~20 identical nucleotides. These results highlight the benefits of using the PathogenID v2.0 resequencing microarray to characterize geographical variants in the follow-up of haemorrhagic fever epidemics; to manage patients and protect communities; and in cases of bioterrorism.

Resequencing microarray method for molecular diagnosis of human arboviral diseases.

BACKGROUND: Resequencing DNA microarray (RMA) technology uses probes designed to identify a panel of viral sequences. It can be used for detecting emerging viruses by revealing the nucleotide polymorphisms within the target of interest. OBJECTIVES/STUDY DESIGN: As a new tool for molecular diagnosis of arbovirus infection, high density PathogenID v2.0 RMA (PID2-RMA) was assessed for the detection and genetic analysis of dengue, West Nile, and Chikungunya viruses in spiked blood samples or sera from individuals infected with dengue virus. Viral RNAs extracted from biological samples were retrotranscribed into cDNA and amplified using the Phi 29 polymerase-based method. This amplified cDNA was used for hybridization on PID2-RMA. RESULTS: A good specificity of RMA-based detection was demonstrated using a panel of arboviruses including Dengue, West Nile and Chikungunya viruses. This technology was also efficient for the detection and genetic analysis of the different serotypes of dengue virus in sera of infected patients. Furthermore, the mixing of dengue, West Nile and Chikungunya prototype viruses within a single sample of human blood did not interfere with the sensitivity of PID2-RMA. CONCLUSIONS: Our data show that high density PID2-RMA was suitable for the identification of medically important arboviruses. It appears to be particularly adapted to the genetic analysis of dengue, West Nile, and Chikungunya viruses in urgent clinical situations where the rapid identification and characterization of the pathogen is essential.

In vitro susceptibility of Aspergillus spp. clinical isolates to albendazole.

The in vitro antifungal activity of albendazole, a benzimidazole widely used as an antihelmintic drug in humans, was investigated and assessed for its activity against Aspergillus spp. Forty-eight isolates, representing the most frequent species found in human pathology [Aspergillus fumigatus (n = 27), Aspergillus flavus (n = 10), Aspergillus terreus (n = 7), Aspergillus nidulans (n = 3) and Aspergillus niger (n = 1)], and one quality control strain (A. niger ATCC 9804 83435) were tested according to the NCCLS M38-P methodology for moulds. All the strains were susceptible to albendazole, with homogeneous MICs for each species; three strains were resistant to itraconazole.

Translesional synthesis on DNA templates containing the 2'-deoxyribonolactone lesion.

A site-specifically modified oligonucleotide containing a single 2'-deoxyribonolactone lesion was used as a template for primer extension reactions catalyzed by M-MuLV reverse transcriptase (RT) and by the Klenow fragments of Escherichia coli DNA polymerase proficient (KF exo(+)) or deficient (KF exo(-)) in exonuclease activity. Analysis of the extension products in the presence of the four dNTPs or of a single dNTP showed that the M-MuLV RT was completely blocked and did not incorporate any dNMP opposite 2'-deoxyribonolactone. KF exo(-) preferentially incorporated nucleotides opposite the lesion following the frequency order dAMP > dGMP >> dTMP approximately dCMP and thus appeared to obey the 'A rule' for preferential incorporation as has been shown previously for the 2'-deoxyribose abasic site. In the sequence context examined, the primer extension by KF exo(-) appeared to be less efficient when dAMP was positioned opposite the lesion as compared with dTMP or dGMP. These two nucleotides promoted a more efficient polymerization accompanied by nucleotide deletion through misalignment incorporations. We therefore predict that the sequence context may strongly influence the translesional synthesis by KF exo(-) and thus the miscoding and mutational potential of the 2'-deoxyribonolactone in E.coli.

The abasic site as a target for generation of locally multiply damaged sites.

Abasic sites in DNA have been specifically targeted by synthetic compounds able to cleave DNA at abasic sites and to induce photodamages in the vicinity of the lesion. The synthesis and the photoactivity of the drugs on abasic sites containing DNA and oligonucleotides are reported.

Search for DNA repair inhibitors: selective binding of nucleic bases-acridine conjugates to a DNA duplex containing an abasic site.

The abasic site is one of the most frequent DNA lesions generated by spontaneous or enzymatic cleavage of the N-glycosidic bond. The abasic site is also an intermediate in the nucleotide and base excision DNA repair. We examined molecules which recognize and cleave DNA at the abasic site with high efficiency. These molecules incorporate in their structure a nucleic base for abasic site recognition, an intercalator for DNA binding, and a polyamino linker for ionic interaction and DNA cleavage. Such compounds, by interfering with abasic sites in DNA, are also inhibitors of DNA repair. In order to better understand the parameters of the interaction, we carried out a UV thermal denaturation study of synthetic oligonucleotides containing the lesion both in the absence and in the presence of the drugs. A similar study was also carried out using the corresponding nonmodified oligonucleotide. The results indicate selective binding of the base-chain-intercalator conjugates to the abasic site containing oligonucleotides.

Solution conformation of an abasic DNA undecamer duplex d(CGCACXCACGC) x d(GCGTGTGTGCG): the unpaired thymine stacks inside the helix.

The three-dimensional structural analysis of DNA undecamer 5'd(C1G2C3A4C5X6C7A8C9G10C11)3', 3'd(G22C21G20T19G18T17G16T15G14C13G12)5' duplex in which the X residue is a modified abasic site [3-hydroxy-2-(hydroxymethyl)tetrahydrofuran] has been performed using NOESY, DQFCOSY, TOCSY, and 31P-1H HSQC-TOCSY spectra in relation with molecular dynamics simulations. A total of 249 distances and 224 dihedral angles were used for construction. The optimal distances were calculated using the complete relaxation matrix method from hybrid matrices which were built with the experimental NOE intensities and additional data derived from either standard A- or B-DNA. Six independent refined structures starting from canonical A- and B-DNA were determined on the basis of the NMR data, and all converged to a single family with average rms deviations below 0.6 A and final NOE Rx factors of 0.055 +/- 0.03. A satisfactory agreement was obtained between measured NOE intensities and those resulting from full relaxation matrix calculations. A single intrahelical form of right-handed DNA duplex is observed; the aromatic base of residue T17 opposite the abasic site is stacked inside the helix. No clear correlation was detected between the C5 and C7 residues, excluding their proximity and the looping out of the abasic site. The abasic site induces a kink of about 30 degrees in the DNA duplex. This kink allows the formation of a bifurcated hydrogen bond between the amino protons of C5 and the O4 oxygen of T17. A detailed analysis of the final structures and their comparison with previous studies of abasic site lesions are described.

Design of molecules that specifically recognize and cleave apurinic sites in DNA.

We have prepared a series of tailor-made molecules that recognize and cleave DNA at apurinic sites in vitro. These molecules incorporate in their structure different units designed for specific function: an intercalator for DNA binding, a nucleic base for abasic site recognition and a linking chain of variable length and nature (including amino and/or amido functions). The cleavage efficiency of the molecules can be modulated by varying successively the nature of the intercalating agent, the nucleic base and the chain. All molecules bind to native calf thymus DNA with binding constants ranging from 10(4) to 10(6) M-1. Their cleavage activity was determined on plasmid DNA (pBR 322) containing 1.8 AP-sites per DNA-molecule. The minimum requirements for cleavage are the presence of the three units, the intercalator, the nucleic base and at least one amino function in the chain. The most efficient molecules cleave plasmid DNA at nanomolar concentrations. Enzymatic experiments on the termini generated after cleavage of AP-DNA suggest a strand break induced by a beta-elimination reaction. In order to get insight into the mode of action (efficiency, selectivity, interaction), we have used synthetic oligonucleotides containing either a true abasic site at a determined position to analyse the cleavage parameters of the synthetic molecules by HPLC or a chemically stable analog (tetrahydrofuran) of the abasic site for high field 1H NMR spectrometry and footprinting experiments. All results are consistent with a beta-elimination mechanism in which each constituent of the molecule exerts a specific function as indicated in the scheme: DNA targeting, abasic site recognition, phosphate binding and beta-elimination catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)