Impact of caspase-1/11, -3, -7, or IL-1ß/IL-18 deficiency on rabies virus-induced macrophage cell death and onset of disease.

Rabies virus is a highly neurovirulent RNA virus, which causes about 59000 deaths in humans each year. Previously, we described macrophage cytotoxicity upon infection with rabies virus. Here we examined the type of cell death and the role of specific caspases in cell death and disease development upon infection with two laboratory strains of rabies virus: Challenge Virus Standard strain-11 (CVS-11) is highly neurotropic and lethal for mice, while the attenuated Evelyn-Rotnycki-Abelseth (ERA) strain has a broader cell tropism, is non-lethal and has been used as an oral vaccine for animals. Infection of Mf4/4 macrophages with both strains led to caspase-1 activation and IL-1ß and IL-18 production, as well as activation of caspases-3, -7, -8, and -9. Moreover, absence of caspase-3, but not of caspase-1 and -11 or -7, partially inhibited virus-induced cell death of bone marrow-derived macrophages. Intranasal inoculation with CVS-11 of mice deficient for either caspase-1 and -11 or -7 or both IL-1ß and IL-18 led to general brain infection and lethal disease similar to wild-type mice. Deficiency of caspase-3, on the other hand, significantly delayed the onset of disease, but did not prevent final lethal outcome. Interestingly, deficiency of caspase-1/11, the key executioner of pyroptosis, aggravated disease severity caused by ERA virus, whereas wild-type mice or mice deficient for either caspase-3, -7, or both IL-1ß and IL-18 presented the typical mild symptoms associated with ERA virus. In conclusion, rabies virus infection of macrophages induces caspase-1- and caspase-3-dependent cell death. In vivo caspase-1/11 and caspase-3 differently affect disease development in response to infection with the attenuated ERA strain or the virulent CVS-11 strain, respectively. Inflammatory caspases seem to control attenuated rabies virus infection, while caspase-3 aggravates virulent rabies virus infection.

Endemicity of leptospirosis in domestic and wild animal species from Reunion Island (Indian Ocean).

Leptospirosis is the major infectious disease on Reunion Island but little is known about the animal reservoir. We conducted a wide-ranging survey that included samples from 574 animals belonging to 12 species. The seroprevalence and prevalence of renal carriage varied greatly depending on the species, with the highest seroprevalence (79·5%) found in Norway rats, and the lowest (13·2%) in tenrecs. The renal carriage rate ranged from 84·6% in mice to 0% in tenrecs. Our results suggest that rodents are the most important reservoirs of leptospirosis on Reunion Island. The epidemiological role that animals play in human infection is discussed. For the first time, we quantified the renal concentration of leptospires in ten naturally infected mammals. The history of Reunion Island colonization probably explains why the circulating Leptospira serogroups were similar to those found in Europe. Our study provides evidence that will help implement preventive measures against this zoonosis.

Pseudomonas aeruginosa outbreak linked to mineral water bottles in a neonatal intensive care unit: fast typing by use of high-resolution melting analysis of a variable-number tandem-repeat locus.

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections in intensive care units. Determining a system of typing that is discriminatory is essential for epidemiological surveillance of P. aeruginosa. We developed a method for the typing of Pseudomonas aeruginosa, namely, multiple-locus variable-number tandem-repeat (VNTR) typing with high-resolution melting analysis (HRMA). The technology was used to genotype a collection of 43 environmental and clinical strains isolated during an outbreak in a neonatal intensive care unit (NICU) that we report. Nineteen strains isolated in other departments or outside the hospital were also tested. The genetic diversity of this collection was determined using VNTR-HRMA, with amplified fragment length polymorphism (AFLP) analysis as a reference. Twenty-five and 28 genotypes were identified, respectively, and both techniques produced congruent data. VNTR-HRMA established clonal relationships between the strains of P. aeruginosa isolated during the outbreak in the NICU and proved, for the first time, the role of mineral water as the inoculum source. VNTR typing with one primer pair in association with HRMA is highly reproducible and discriminative, easily portable among laboratories, fast, and inexpensive, and it demonstrated excellent typeability in this study. VNTR-HRMA represents a promising tool for the molecular surveillance of P. aeruginosa and perhaps for molecular epidemiologic analysis of other hospital infections.

Simultaneous detection and quantitation of Chikungunya, dengue and West Nile viruses by multiplex RT-PCR assays and dengue virus typing using high resolution melting.

Chikungunya (CHIKV), Dengue (DENV) and West Nile (WNV) viruses are arthropod-borne viruses that are able to emerge or re-emerge in many regions due to climatic changes and increase in travel. Since these viruses produce similar clinical signs it is important for physicians and epidemiologists to differentiate them rapidly. A molecular method was developed for their detection and quantitation in plasma samples and a DENV typing technique were developed. The method consisted in performing two multiplex real-time one-step RT-PCR assays, to detect and quantify the three viruses. Both assays were conducted in a single run, from a single RNA extract containing a unique coextracted and coamplified composite internal control. The quantitation results were close to the best detection thresholds obtained with simplex RT-PCR techniques. The differentiation of DENV types was performed using a High Resolution Melting technique. The assays enable the early diagnosis of the three arboviruses during viremia, including cases of coinfection. The method is rapid, specific and highly sensitive with a potential for clinical diagnosis and epidemiological surveillance. A DENV positive sample can be typed conveniently using the High Resolution Melting technique using the same apparatus.

Begomovirus 'melting pot' in the south-west Indian Ocean islands: molecular diversity and evolution through recombination.

During the last few decades, many virus species have emerged, often forming dynamic complexes within which viruses share common hosts and rampantly exchange genetic material through recombination. Begomovirus species complexes are common and represent serious agricultural threats. Characterization of species complex diversity has substantially contributed to our understanding of both begomovirus evolution, and the ecological and epidemiological processes involved in the emergence of new viral pathogens. To date, the only extensively studied emergent African begomovirus species complex is that responsible for cassava mosaic disease. Here we present a study of another emerging begomovirus species complex which is associated with serious disease outbreaks in bean, tobacco and tomato on the south-west Indian Ocean (SWIO) islands off the coast of Africa. On the basis of 14 new complete DNA-A sequences, we describe seven new island monopartite begomovirus species, suggesting the presence of an extraordinary diversity of begomovirus in the SWIO islands. Phylogenetic analyses of these sequences reveal a close relationship between monopartite and bipartite African begomoviruses, supporting the hypothesis that either bipartite African begomoviruses have captured B components from other bipartite viruses, or there have been multiple B-component losses amongst SWIO virus progenitors. Moreover, we present evidence that detectable recombination events amongst African, Mediterranean and SWIO begomoviruses, while substantially contributing to their diversity, have not occurred randomly throughout their genomes. We provide the first statistical support for three recombination hot-spots (V1/C3 interface, C1 centre and the entire IR) and two recombination cold-spots (the V2 and the third quarter of V1) in the genomes of begomoviruses.

A New Tomato leaf curl virus from Mayotte.

In June 2003, symptoms of stunting and leaf curling resembling symptoms of tomato leaf curl disease, as well as reductions in yields, were observed on tomato plants in the western (Combani and Kahani) and eastern (Dembeni, Kaoueni, and Tsararano) regions of Mayotte, a French island in the Comoros Archipelago located in the northern part of the Mozambique Channel. The whitefly, Bemisia tabaci (Gennadius), was observed colonizing tomato plants and other vegetable crops at low levels. Overall, 13 leaf samples with symptoms were collected from tomato plants among the five regions and tested for the presence of begomoviruses using a polymerase chain reaction (PCR) assay with two sets of degenerate primers designed to amplify two regions of the A component of begomoviruses. Primers MP16 and MP82 amplify an approximately 500-bp fragment located between the intergenic conserved nonanucleotide sequence and the first 200 bp of the coat protein (CP) gene (2). Primers AV494 and AC1048 amplify the approximately 550-bp core region of the CP gene (3). Six leaf samples, one from Combani, three from Dembeni, and two from Kahani, gave a PCR product of the expected size with both sets of primers. No PCR products were obtained with degenerate primers designed for begomovirus DNA B or ß. The approximately 500- and 550-bp PCR products from one sample each of Combani (EMBL Accession Nos. AJ620912 and AJ620915, respectively), Dembeni (EMBL Accession Nos. AJ620911 and AJ620914, respectively), and Kahani (EMBL Accession Nos. AJ620913 and AJ620916, respectively) were sequenced. For the 489-bp sequences obtained with the MP16/MP82 primer set, the three isolates had 90 to 95% nucleotide identity (DNAMAN; Lynnon BioSoft, Quebec). The most significant sequence alignments (NCBI and BLAST) were with begomoviruses; 80 to 83% nucleotide identity was obtained with the Tomato yellow leaf curl Morondava virus (TYLCMV) isolates from Madagascar (EMBL Accession Nos. AJ422123 and AJ422124), 80 to 82% nucleotide identity was obtained with the South African cassava mosaic virus (SACMV) isolates (GenBank and EMBL Accession Nos. AF155806 and AJ422132), and 79 to 81% nucleotide identity was obtained with the East African cassava mosaic Malawi virus (EMBL Accession No. AJ006460). For the 522-bp sequences obtained with the AV494/AC1048 primer set, 95 to 97% nucleotide identity was shown between the three isolates. The most significant sequence alignments were also with begomoviruses; TYLCMV isolate Morondava (EMBL Accession No. AJ422125) with 86 to 88% nucleotide identity, Tomato yellow leaf curl virus isolates (GenBank and EMBL Accession Nos. AF105975, AJ489258, AB014346, AF024715, AF071228, and X76319) with 86 to 87% nucleotide identity, and SACMV isolate M12 (EMBL Accession No. AJ422132) with 85 to 86% nucleotide identity. According to the current taxonomic criteria for the provisional classification of a new begomovirus species, nucleotide sequence identity in the core region of the CP <90% (1), the tomato begomovirus from Mayotte is a new species and is provisionally named Tomato leaf curl Mayotte virus. References: (1) J. K. Brown et al. Arch. Virol. 146:1581, 2001. (2) P. Umaharan et al. Phytopathology 88:1262, 1998. (3) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.