Éric Leblanc

Global Genetic Diversity of Human Metapneumovirus Fusion Gene

We analyzed 64 human metapneumovirus strains from eight countries. Phylogenetic analysis identified two groups (A and B, amino acid identity 93%–96%) and four subgroups. Although group A strains predominated, accounting for 69% of all strains, as many B as A strains were found in persons >3 years of age.

Human bocavirus infections in hospitalized children and adults.

The pathogenic role of this virus in infected children is unclear.

Distribution and Clinical Impact of Human Respiratory Syncytial Virus Genotypes in Hospitalized Children over 2 Winter Seasons

Sequencing studies of the glycoprotein G gene were performed in human respiratory syncytial virus (hRSV) strains detected by reverse-transcription polymerase chain reaction directly from nasopharyngeal aspirates of hospitalized children < or =3 years old over 2 winters. Clinical data were compared between 106 children infected with group A hRSV (96 GA2 genotypes) and 94 children infected with hRSV group B (62 GB3 genotypes). A severity index was defined by assigning 1 point each for the use of >30% supplemental oxygen, admission to an intensive-care unit, and duration of hospital stay of >5 days. Group A and genotype GA2 strains were associated with greater severity of hRSV disease than were group B strains.

The role of reduced pterins in resistance to reactive oxygen and nitrogen intermediates in the protozoan parasite Leishmania.

During its life cycle, the protozoan parasite Leishmania experiences oxidative stress when interacting with macrophages. Reduced pterins are known scavengers of reactive oxygen and nitrogen intermediates. Leishmania has a pteridine reductase, PTR1, whose main function is to provide reduced pterins. We investigated the role of PTR1 in resistance to oxidative and nitrosative stress in Leishmania tarentolae, Leishmania infantum, and Leishmania major PTR1(-/-) mutants. The PTR1(-/-) cells of the three species were more sensitive to H2O2- and NO-induced stress. Using a fluorescent probe allowing ROI quantification, we demonstrated an increase in intracellular oxidant molecules in the PTR1(-/-) mutants. The disruption of PTR1 increased metacyclogenesis in L. infantum and L. major. We purified metacyclic parasites from PTR1(-/-) mutants and control cells and tested their intracellular survival in the J774 mouse cell line and in human monocyte-derived macrophages. Our results showed that PTR1(-/-) null mutants survived less in both macrophage models compared to control cells and this decrease was more pronounced in macrophages activated for oxidant production. This study demonstrates that one physiological role of reduced pterins in Leishmania is to deal with oxidative and nitrosative species, and a decreased ability to provide reduced pterins leads to decreased intracellular survival.

Infections by human coronavirus-NL in hospitalized children.

Background: A new human coronavirus (HCoV), HCoV-NL, was recently reported for Dutch patients with acute respiratory tract infections (ARTI). Little information is available on the incidence, clinical manifestations and epidemiologic features of HCoV-NL infections. Methods: We performed a prospective study of symptomatic (case subjects with ARTI) and asymptomatic (control subjects undergoing elective surgery) children, ≤3 years of age, hospitalized in Canada during 2 winter seasons (2001–2003), to look at the prevalence of respiratory viruses. Reverse transcription-PCR assays were used to retrospectively detect HCoV-NL and to correlate its presence with clinical symptoms. Results: HCoV-NL was detected in nasopharyngeal aspirates from 3.0% of young children (12 of 396 children) hospitalized for treatment of ARTI (case subjects), compared with 1.7% of asymptomatic control subjects (3 of 177 children) (P = 0.6). Nine (75.0%) of the symptomatic children had mixed viral infections. The mean age and mean duration of hospitalization of case subjects were 10.1 months and 4.9 days, respectively. Final diagnoses consisted of bronchiolitis or bronchitis (9 of 12 cases), pneumonitis (1 of 12 cases) and upper respiratory tract infections (2 of 12 cases), although 2 of 3 subjects with single HCoV-NL infections had upper respiratory tract infections only. Sequence analysis of the 1a and spike genes revealed that multiple HCoV-NL strains circulated in the same geographical area in each of the 2 winter seasons. Variability was more pronounced for the spike gene, with 2 clusters of strains. Conclusions: HCoV-NL was not a major respiratory pathogen in Canada during our study, as shown by its low detection rate in hospitalized children with ARTI, coupled with the high frequency of additional pathogens and its occasional detection in healthy children.

Human parainfluenza type 4 infections, Canada.

During the fall/winter season of 2004–05, we found 9 respiratory specimens positive for human parainfluenza virus type 4 (HPIV-4) in our laboratory (43% of all HPIVs) from patients with mild to moderate respiratory illnesses. Sequencing studies identified 8 different HPIV-4A strains and 1 HPIV-4B strain.

Plastic Polymers for Efficient DNA Microarray Hybridization: Application to Microbiological Diagnostics

ABSTRACT Fabrication of microarray devices using traditional glass slides is not easily adaptable to integration into microfluidic systems. There is thus a need for the development of polymeric materials showing a high hybridization signal-to-background ratio, enabling sensitive detection of microbial pathogens. We have developed such plastic supports suitable for highly sensitive DNA microarray hybridizations. The proof of concept of this microarray technology was done through the detection of four human respiratory viruses that were amplified and labeled with a fluorescent dye via a sensitive reverse transcriptase PCR (RT-PCR) assay. The performance of the microarray hybridization with plastic supports made of PMMA [poly(methylmethacrylate)]-VSUVT or Zeonor 1060R was compared to that with high-quality glass slide microarrays by using both passive and microfluidic hybridization systems. Specific hybridization signal-to-background ratios comparable to that obtained with high-quality commercial glass slides were achieved with both polymeric substrates. Microarray hybridizations demonstrated an analytical sensitivity equivalent to approximately 100 viral genome copies per RT-PCR, which is at least 100-fold higher than the sensitivities of previously reported DNA hybridizations on plastic supports. Testing of these plastic polymers using a microfluidic microarray hybridization platform also showed results that were comparable to those with glass supports. In conclusion, PMMA-VSUVT and Zeonor 1060R are both suitable for highly sensitive microarray hybridizations.

Divergence among Genes Encoding the Elongation Factor Tu of Yersinia Species

Elongation factor Tu (EF-Tu), encoded by tuf genes, carries aminoacyl-tRNA to the ribosome during protein synthesis. Duplicated tuf genes (tufA and tufB), which are commonly found in enterobacterial species, usually coevolve via gene conversion and are very similar to one another. However, sequence analysis of tuf genes in our laboratory has revealed highly divergent copies in 72 strains spanning the genus Yersinia (representing 12 Yersinia species). The levels of intragenomic divergence between tufA and tufB sequences ranged from 8.3 to 16.2% for the genus Yersinia, which is significantly greater than the 0.0 to 3.6% divergence observed for other enterobacterial genera. We further explored tuf gene evolution in Yersinia and other Enterobacteriaceae by performing directed sequencing and phylogenetic analyses. Phylogenetic trees constructed using concatenated tufA and tufB sequences revealed a monophyletic genus Yersinia in the family Enterobacteriaceae. Moreover, Yersinia strains form clades within the genus that mostly correlate with their phenotypic and genetic classifications. These genetic analyses revealed an unusual divergence between Yersinia tufA and tufB sequences, a feature unique among sequenced Enterobacteriaceae and indicative of a genus-wide loss of gene conversion. Furthermore, they provided valuable phylogenetic information for possible reclassification and identification of Yersinia species.

Antifolate Resistance Mechanisms from Bacteria to Cancer Cells with Emphasis on Parasites

Reduced folates serve as co-factors in a variety of one-carbon transfer reactions including the biosynthesis of thymidylate, of purine nucleotides, and of the amino acids serine and methionine. Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) catalyze consecutive reactions in the de novo synthesis of dTMR In protozoa and plants, these two enzymes are fused resulting in a DHFR-TS protein (Ferone and Roland, 1980). The enzyme DHFR is the target for the action of antifolates, which are widely used during chemotherapeutic interventions. Commonly used antifolates are shown in Figure 1. The folate antagonist trimethoprim (TMP) is used to treat bacterial infections caused by urinary tract or enteric pathogens (Huovinen et al., 1995). The antifolate pyrimethamine (PYR) is used against the protozoan parasites Plasmodium and Toxoplasma (Borst and Ouellette, 1995) whereas trimetrexate with leucovirin is used in fungal infections caused by Pneumocystis carinii (Hitchings, 1989). The antifolate methotrexate (MTX) is widely used for the treatment of various forms of cancer as well as for the treatment of rheumatoid arthritis, psoriasis and some autoimmune diseases (Gorlick et al., 1996). The primary structures of DHFR from a variety of organisms share very little homology, hence explaining, in part, the specificity and activity of different antifolates against targeted organisms. The basis for this selectivity was studied further by using comparative enzymology and structural analyses. Differences in the kinetic properties of the various DHFRs are likely to contribute to the selectivity of antifolates (Schweitzer et al., 1990).

Rapid Filtration Separation-Based Sample Preparation Method for Bacillus Spores in Powdery and Environmental Matrices

ABSTRACT Authorities frequently need to analyze suspicious powders and other samples for biothreat agents in order to assess environmental safety. Numerous nucleic acid detection technologies have been developed to detect and identify biowarfare agents in a timely fashion. The extraction of microbial nucleic acids from a wide variety of powdery and environmental samples to obtain a quality level adequate for these technologies still remains a technical challenge. We aimed to develop a rapid and versatile method of separating bacteria from these samples and then extracting their microbial DNA. Bacillus atrophaeus subsp. globigii was used as a simulant of Bacillus anthracis. We studied the effects of a broad variety of powdery and environmental samples on PCR detection and the steps required to alleviate their interference. With a benchmark DNA extraction procedure, 17 of the 23 samples investigated interfered with bacterial lysis and/or PCR-based detection. Therefore, we developed the dual-filter method for applied recovery of microbial particles from environmental and powdery samples (DARE). The DARE procedure allows the separation of bacteria from contaminating matrices that interfere with PCR detection. This procedure required only 2 min, while the DNA extraction process lasted 7 min, for a total of <10 min. This sample preparation procedure allowed the recovery of cleaned bacterial spores and relieved detection interference caused by a wide variety of samples. Our procedure was easily completed in a laboratory facility and is amenable to field application and automation.